Magnetic Nanoparticles with Covalently Bound Self-Assembled

Article pubs.acs.org/JPCC

Magnetic Nanoparticles with Covalently Bound Self-Assembled Protein Corona for Advanced Biomedical Applications Rina 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, 771 47 Olomouc, Czech Republic ‡

S Supporting Information *

ABSTRACT: Novel surface active maghemite nanoparticles (SAMNs) possessing peculiar colloidal properties and surface characteristics are able to covalently bind biomolecules. The interactions of SAMNs and rhodamine derivatized SAMNs (SAMN@RITC) with proteins from cell culture medium were studied by gel electrophoresis and mass spectrometry. Among the 3000 proteins present in fetal calf serum, SAMNs and SAMN@RITC give rise to the formation of a self-assembled corona shell with 22 selected proteins, representing minor plasma proteins, among which α-2-HS- glycoprotein stands out. Bovine serum albumin (BSA), representing 80% of the total serum proteins, shows negligible absorption on the SAMN surface. Nevertheless, SAMNs are able to bind BSA, upon incubation in pure BSA solutions. The interaction between SAMNs and BSA was investigated by optical spectroscopy, circular dichroism, Fourier transform infrared spectroscopy, and transmission electron microscopy. BSA binding resulted a time-consuming process, nevertheless experimental results showed the interaction of 6 ± 2 BSA molecules per nanoparticle, and optical spectra indicate remarkable changes in SAMN optical features, as well as circular dichroism proved secondary structure alteration of bound BSA, suggesting that the protein needs to adapt its structure to adhere to nanoparticle surface. The selectively bound protein corona shell, formed upon SAMNs incubation in calf serum, was responsible for the characteristic behavior when SAMNs were tested for cell internalization and cytotoxicity on HeLa cells. Cytotoxicity of SAMN preparations was extensively studied, and was negligible up to 100 μg mL−1. Moreover, nanoparticle uptake proceeded for long times, 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 based nanobiocomposites can be employed for the preparation of self-assembled opsonized nanoparticles as future candidates for biomedical applications.

1. INTRODUCTION Many different kinds of magnetic nanoparticles have already demonstrated their potential in biomedical applications,1,2 as these nanostructures properly labeled with bioactive molecules can serve in magnetic separations,3−5 drug delivery systems,6 or to generate heat by exposition to an alternating electromagnetic field, thus increasing the temperature of tumor tissues and destroying pathological cells.7 Moreover, their use in magnetic resonance imaging as contrast agents is common,8,9 owing to their unique magnetic properties and biocompatibility,10 and commercial preparations are available (Nanocs Inc., New York, NY; Nanoimmunotech SL, Vigo, Spain; MK Impex Corp., Missisauga, ON, Canada; and many others). However, for most iron oxide nanoparticles, little is known about their potential adverse effects on health due to prolonged exposure in biological systems, representing a hindrance to the develop© 2013 American Chemical Society

ment of novel applications of magnetic nanoparticles in nanobiology, nanomedicine, and nanotoxicology. The metabolic and immunological responses induced by these particles have been rarely understood so far.11 An essential prerequisite for the implementation of bionanotechnological applications is to obtain nanoparticles with a hydrophilic surface able to maintain colloidal stability under physiological conditions.12 Among magnetic nanoparticles, magnetite (Fe3O4) appears to be an interesting candidate, owing to its low toxicity, high saturation magnetization, and susceptibility. Unfortunately, upon exposure to physiological environments, magnetite nanoparticles exhibit a Received: July 10, 2013 Revised: August 24, 2013 Published: September 5, 2013 20320

dx.doi.org/10.1021/jp4068137 | J. Phys. Chem. C 2013, 117, 20320−20331

The Journal of Physical Chemistry C

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glucose oxidase.22 In both cases, immobilized enzymes on the nanomaterial retained their catalytic activity. In the present work, we combined the advantages of immobilized fluorescent probes on nanomaterials and the easy drivability of magnetic nanoparticles to propose a low toxicity system for cells, providing a novel bimodal nanodevice, which can be incorporated in cells, for future applications in targeted drug delivery and theranostics.

tendency to aggregate, which considerably reduces their efficiency, and more importantly, the magnetite surface tends to oxidize, modifying its properties. Alternatively, maghemite nanoparticles appear very promising in theranostic applications.13 However, obtaining monodisperse, colloidal suspensions of maghemite nanoparticles in aqueous media is a challenging task, due to the complexity of controlling the nucleation and growth processes in water. Recently, we developed a new simple wet method to synthesize superparamagnetic nanoparticles constituted of stoichiometric maghemite (γ-Fe2O3) in the dimension range around 10 nm, obtaining monodisperse preparations in aqueous media.14,15 This new category of magnetic nanoparticles 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 colloidal suspensions, present a high average magnetic moment and can be easily derivatized to immobilize specific organic molecules in solution. SAMNs were already functionalized with ligands of high biotechnological interest, such as biotin and avidin, by simple incubation in aqueous solution.3 Nevertheless, the study of SAMN interactions with proteins is still meaningful, and their effect on protein function and structure is worth for further studies. Furthermore, the investigation of protein adsorption and interactions at interfaces is important from a medical, biotechnological, and industrial point of view.16 The present report describes a detailed investigation on SAMNs binding to a model biological host, namely bovine serum albumin (BSA), leading to a stable complex (SAMN@ BSA). BSA has been one of the most extensively studied proteins, particularly for its structural homology with human serum albumin.17,18 It is a single polypeptide chain protein of 66 kDa, which is cross-linked by 17 disulfide bonds.19,20 The SAMN@BSA complex was prepared and studied with different spectroscopic technique and transmission electron microscopy. Therefore, the behavior of SAMNs was studied in physiological cell culture media, evidencing the high nanoparticle colloidal stability under physiological conditions. Protein absorption on SAMNs, mimicking in vivo opsonization, was analyzed by mass spectrometry, showing that SAMNs interact selectively with different minor serum proteins, among which α-2-HSglycoprotein stands out. These results evidenced that SAMNs display specific binding behavior toward protein molecules and that their behavior in real systems is not easily predictable. Furthermore, we studied SAMNs cytotoxicity and their effect on HeLa cells, demonstrating that they can be safely used at concentrations up to 100 μg mL−1. Moreover, these unique maghemite nanoparticles, exhibiting excellent colloidal behavior without any additional organic or inorganic surface coating, were exploited for the immobilization of rhodamine B isothiocyanate (RITC) acting as a fluorescent label and, at the same time, as spacer arm, useful to immobilize enzymes.21,22 As a result, a magnetically drivable rhodaminebased fluorescent nanocomposite has been synthesized. In our previous works we demonstrated that SAMNs@RITC represents a friendly environment for immobilized enzymes, and we proposed this fluorescent magnetically drivable nanocarrier as an universal tool for creating fluorescent magnetic drivable enzymatic nanosystems. We already reported on two different enzymes used to prepare magnetic drivable, fluorescent nanocatalysts: bovine serum amine oxidase21 and

2. EXPERIMENTAL SECTION 2.1. Materials. Chemicals were purchased at the highest commercially available purity and were used without further treatment. Iron(III) chloride hexahydrate (97%), sodium borohydride (NaBH4), rhodamine B isothiocyanate (RITC), tetramethylammonium perchlorate, and ammonium hydroxide solution (35% in water) were obtained from Sigma-Aldrich (Italy), as well as trypan blue, resazurin, and RNase. HeLa cells were form ATCC (Middlesex, U.K.). Sterile filtered fetal bovine serum (FBS) was from Sigma-Aldrich (cod. F9665). Propidium iodide, tetramethyl-rhodamine, CM-H2DCFDA (general oxidative stress indicator), MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide), penicillin (10 000 U mL−1), streptomycin (10 000 μg mL−1), glutamine solutions, and acetomethoxy calcein (calcein AM) were from Molecular Probes-Invitrogen (U.K.). Buffers were prepared according to standard laboratory procedures using Milli-Q reagent grade water (Merck Millipore, Billerica, MA). Intrumentation and methods are reported in the Supporting Information. 3. RESULTS 3.1. SAMN Surface Functionalization with BSA. SAMN surface functionalization was accomplished with BSA by simple incubation of reactants in 50 mM tetramethylammonium perchlorate, at pH 7.0. In particular, SAMN colloidal dispersions (50 mg L−1) were incubated with BSA (100 mg L−1) under overnight end-over-end mixing at 4.0 °C. It should be noticed that, in our previous work on SAMN@avidin preparations, the binding process occurred within less than 1 h, preserving avidin biological activity upon immobilization on SAMN surface, and suggesting that protein tertiary structure was unaltered.3 Conversely, in the present case, BSA binding resulted negligible for short incubation times (1 h) and the incubation period was prolonged overnight. After the incubation period, nanoparticles were separated by the application of an external magnetic field and the presence of the biomolecule in the supernatant was checked by spectrophotometry. SAMN@BSA complex was magnetically isolated and washed several times with 50 mM tetramethylammonium perchlorate, pH 7.0, and water. Biomolecule coverage on SAMNs was stable, without any loss in solution of BSA, as checked by spectrophotometry. UV−vis absorption is a simple and easily applicable technique to explore structural changes on the surface of nanomaterials and to prove complex formations.3 The electronic absorption spectrum of bare SAMNs, acquired in water, shows a wide band with a maximum at about 400 nm (Figure S1 in the Supporting Information) characterized by an extinction coefficient of 1520 M−1 cm−1, expressed as Fe2O3 molar concentration. The interaction of BSA with SAMN surface produced an alteration of nanoparticle optical properties (see Figure S1). Upon BSA binding, both the shape and 20321



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

position of maximum absorption were altered with respect to bare SAMNs, confirming the influence of SAMN surface properties on optical characteristics, as already extensively described in our previous work.3 In water, the spectrum of SAMN@BSA is characterized by a peak at 436 and a shoulder at 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 nanoparticle concentration, in the range 200−1800 mg L−1, in 50 mM tetramethylammonium perchlorate, pH 7.0. Experimental data showed the presence of 6 ± 2 BSA molecules per nanoparticle, corresponding to 3 ± 1 × 10−2 mg of BSA per milligram of nanoparticles, in agreement with previous work on avidin binding.3 BSA molecules, in water, have a prolate ellipsoidal shape, with the axes being 11.6 and 2.7 nm.23 As a comparison, the possible theoretical number of BSA molecules on SAMN surface, considering proteins positioned on the SAMN surface along their 11.6 nm axis, gives 10 BSA molecules per nanoparticle. The Fourier transform infrared (FTIR) spectra of SAMN@ BSA show the presence of typical protein IR bands, attributable to vibrational frequencies of C−C, C−O, and CH2− groups, in the 1000−1700 cm−1 range, confirming the presence of BSA bound on nanoparticle surface (see Figure 1). In particular, the characteristic protein amide I and amide II IR bands at 1654 and 1544 cm−124 are present in the SAMN@BSA FTIR spectrum. SAMN@BSA was also studied by transmission electron microscopy (TEM) and FTIR. TEM microscopy images of the SAMN@BSA complex, reported in Figure 2, indicate the presence of an organic matrix shell, of about 3 nm thickness around the iron oxide nanoparticles, characterized by a lower electron density. The aggregation phenomenon showed by the coated nanoparticles is probably due to TEM sample pretreatment and drying on the copper grid. In water suspensions SAMN@BSA still maintain their stable colloidal behavior. The shell thickness is in good agreement with BSA short axis (2.7 nm),23 suggesting that the protein adheres to SAMN surface by laying on its long axis. Considering that BSA

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

long axis is 11.6 nm, thus, protein should bend to adapt itself to nanoparticle curvature, and as a consequence causing an alteration of its tertiary structure. In fact, it is well-known by literature that the interactions between proteins and solid surfaces could lead to significant changes in protein structure,25,26 possibly altering protein functionality and biological activity. Conformational changes on BSA induced by SAMN binding were detected using circular dichroism spectroscopy (CD). CD spectra of BSA and SAMN@BSA are reported in Figure 3. CD spectrum of BSA resulted consistent with a previously published measurements,27 while the spectrum of BSA adsorbed onto SAMN surface was markedly altered, confirming the interaction occurrence. Native BSA clearly shows a sharp absorption at 190 nm and two negative absorption bands: one is attributed to carbonyl excitation in polypeptide chains due to π−π* transition at 209 nm, and another one at 222 nm corresponding to an n−π* transition.28 The characteristic positive sharp peak at 190 nm and negative double 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 peaks similar to those observed upon binding on silica coated nanoparticles27 confirming a substantial alteration in α-helical 20322



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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 mM potassium 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 the average of 16 scans. (black line) BSA; (red line) SAMN@BSA.

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

score

TSP1 TETN FETUA KNG1 APO-B100 APOA1 APO-E

thrombospondin-1 tetranectin α-2-HS-glycoprotein kininogen-1 apolipoprotein-B100 apolipoprotein-A1 apolipoprotein E

1280 1185 841 188 1335 617 430

PPIB GPX3 CO3 CFAB

peptidyl-prolyl isomerase B glutathione peroxidase 3 complement C3 complement factor B

381 384 658 294

CO9 HBBF HBA A1AT SPP24

complement C9 fetal hemoglobin sub. β hemoglobin sub. α α-1-antiproteinase secreted phospho-prot.24

365 920 274 571 545

MYH9 ALBU HSPB1

myosin-9 serum albumin heat shock protein beta-1

1093 755 536

SAMN@RITC

Score

Total removed proteins (NH4OH)

gel band

TSP1 TETN FETUA KNG1 APO-B100 APOA1 APO-E APOA2 PPIB GPX3 CO3 CFAB CFAH

thrombospondin-1 tetranectin α-2-HS-glycoprotein kininogen-1 apolipoprotein-B100 apolipoprotein-A1 apolipoprotein E apolipoprotein-A2 peptidyl-prolyl isomerase B glutathione peroxidase 3 complement C3 complement factor B complement factor H

1088 916 1081 590 1335 1116 550 285 381 384 201 672 334

3 11 7, 8

HBBF HBA A1AT SPP24 A2MG MYH9 ALBU HSPB1

fetal hemoglobin sub. β hemoglobin sub. α α-1-antiproteinase secreted phospho-prot.24 α-2-macroglobulin myosin-9 serum albumin heat shock protein beta-1

1694 1031 722 457 458 1250 546 536

yes yes yes yes no yes yes yes no no yes no yes yes yes yes yes yes yes yes yes no

1 9 13 10 10 4 5 4 6 12 12 7 11 2 6 10

a

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

positioned anchor groups on avidin for a multiple point binding on SAMN surface, while BSA needs to adapt itself for a successful absorption. This second process reasonably takes more time and causes dramatic structure modifications. 3.2. Mass Spectrometry Analysis of Protein Bound to SAMNs. It is well-known that iron oxide nanoparticles are able to bind plasma proteins and that proteins tend to coat the

structure of the protein in direct contact with surface groups on the magnetic particles. Differently from the case of avidin binding, occurring in short times (1 h) and without protein structure alterations,3 the binding of BSA to SAMNs was time-consuming (at least 12 h), leading to dramatic changes in the protein tertiary structure. A possible explanation can be due to the presence of properly 20323



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nanoparticle surface upon their entrance into biological environments.29,30 Moreover, the function and fate of nanoparticles in biological systems are not only related to the properties of nanoparticle surface, but also to bound proteins.31,32 Actually, living cells recognize the protein modified surface of nanoparticles. Generally, bare or functionalized iron oxide nanoparticles reported in literature are able to absorb serum proteins exclusively by electrostatic interactions.33 Conversely, bare SAMNs bind molecules by complexation to undercoordinated iron(III) sites or, in the case of SAMN@RITC, by nucleophilic addition to the isothiocynate moiety. As a consequence, no significant contribution of electrostatic interactions should be expected 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 bovine serum (FBS) and the amount and nature of absorbed proteins on nanoparticle surface was determined by gel electrophoresis and mass spectrometry. Considering that temperature could influence the formation of a protein corona on nanoparticle surface when incubated in cell culture medium,34 in the present case 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), proteins associated with nanoparticle surface were released by incubation in 0.5 M ammonium hydroxide, which was already tested for the removal of bound molecules from SAMN surface,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 Fisher Scientific Inc., Rockford, IL). Bound proteins resulted 42.4 and 82.8 mg g−1 nanoparticles in the case of bare SAMNs and SAMN@RITC, respectively, in agreement with previous studies on avidin binding on SAMNs.3 Protein samples removed by ammonium hydroxide were dried at reduced pressure, resuspended in 40 mM ammonium bicarbonate, containing of 10% acetonitrile, and were digested overnight at 37 °C by trypsin treatment. The resulting peptides were 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/SDS buffer and analyzed by gel electrophoresis, along with samples obtained from protein removal by TRIS/SDS treatment. Most intense electrophoretic bands (see Figure 4) were excised and digested by trypsin, and proteins were identified by mass spectrometry as above (see “gel band” column in Table 1). Notwithstanding SAMNs and SAMN@RITC have different surface chemistries, electrophoretic profiles of proteins removed from both these nanoparticles are quite similar (Figure 4). Indeed, as reported in Table 1, among the 22 identified plasma proteins, only 4 were not detected in both SAMNs and SAMN@RITC, and none of these proteins represents a major serum component. This suggests that the presence of available anchoring groups on protein structure is necessary but not sufficient for effective binding. Proteins should present a proper conformation in order to guarantee an efficient multiple point binding on SAMNs surface, and, as consequence, protein tertiary structure should play a main role in discriminating protein binding efficiency. In addition, the small number of protein associated with SAMNs (only 22 proteins, among about 3000 identified in plasma, as reported by Omenn et al.35) indicates that protein binding to nanoparticle

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

surface is selective. Bound proteins (Table 1) can be divided into six main groups: histidine-rich glycoproteins (thrombospondin, fetuin-A, tetranectin, and kininogen); various apolipoproteins and associated proteins (apo-B100, Apo A1, Apo A2, Apo-E PPIB, and GPX3); proteins belonging to the complement system (C3, C9, and factors B and H); hemoglobin subunits α and β; protease inhibitors (α1antiproteinase, SPP24, and α2-macroglobulin) and the not functionally related BSA, myosin-9, and HSPB1. The most abundant protein, evidenced by intensity in the electrophoretic profile, was α-2-HS-glycoprotein, followed by hemoglobin subunits, thrombospondin, myosin-9 and Apo-B100. The relative protein abundance in FBS does not seem to play a major role in the proteins association with SAMNs. In fact, while α-2-HS-glycoprotein and Apo-B100 are present in FBS in the milligrams per milliliter range (≈15 and 1 mg mL−1, respectively), thrombospondin, myosin-9, and hemoglobin subunits are at the micrograms per milliliter level (between 5 and 40 μg mL−1). Moreover, BSA, which is the most abundant FBS protein (≈25 mg mL−1), resulted to poorly bind on SAMNs (see band 6 in Figure 4) and transferrin, the third plasma protein for abundance (2−3 mg mL −1 ), was undetectable. These results recall the comparison between avidin and BSA binding on SAMNs: the first one tends to bind efficiently, in short times, possibly because it does not need to alter its tertiary structure for stable multiple point binding on SAMN surface. Differently, BSA binding requires long times, probably because the protein must modify its structure to adapt 20324



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to nanoparticle surface. As in the case avidin, the 22 adsorbed medium proteins coat efficiently on SAMNs and SAMN@ RITC surface in a short time, despite the competition with other 3000 kinds of proteins, some of them (BSA) highly concentrated. It is noteworthy that Mahmoudi et al. reported on transferrin interaction with magnetite SPIONs, involving irreversible denaturation,26 working in a system devoid of competition 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 efficient protein removal from SAMN surface than ammonium hydroxide. Data reported in Table 1 shows that SAMN dissociation of Apo-B100, PPIB, GPX3, CFAB, and HSPB1 occurred only in SDS containing buffer. SDS is a powerful protein denaturing agent, whose interactions with polypeptides lead to the complete loss of tertiary structures. Thus, it is conceivable that high affinity interactions with SAMNs depend on specific tertiary structure of the polypeptide amino acid sequence. Clearly, our observations suggest the coexistence of several types of interactions between proteins and SAMNs, attributable to multiple point binding,21 and deserving in-depth investigations. On the other hand, a correlation between protein tertiary structure and affinity for the nanoparticle surface is pointed out. In the present case, a shell of biologically recognizable proteins was formed on the nanoparticle surface, suggesting that, as a consequence of the spatially governed multiple point binding, the best absorbable proteins are those presenting correctly positioned anchoring groups and which do not 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@RITC complexes for biomedical applications, the interaction, internalization processes and cytotoxicity of both the preparations were evaluated on HeLa cell cultures. Hela cells were incubated with bare and RITC coated SAMNs (30 μg mL−1) and analyzed at different times by confocal fluorescence microscopy, which allows the accurate signal selection from specific focal planes and, consequently, the precise fluorescence localization within the cells. The serial acquisition of different planes along the Z axis permitted the reconstruction of three-dimensional distribution of the fluorescence signal. In the present study, in addition to the red fluorescence signal of RITC associated with 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 at 515−530 nm). In fact, HeLa cells were incubated with calcein methyl ester few minutes before the observations. This derivative of calcein was used due to its ability to freely permeates cell membranes, and therefore to spread in all cellular compartments, where, as a result of esterase hydrolysis, becomes fluorescent. Calcein is an excellent marker of cell volume and is useful to precisely define the cell membrane boundary.36 Thus, the colocalization with calcein allows the precise identification of nanoparticles position within the cells. Furthermore, acidic cell compartments, such as lysosomes, are highlighted by negative staining since the fluorescence of calcein 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 HeLa cells, where they appear with various degrees of aggregation (panels 1 and 3). Most of SAMN associated fluorescence

Figure 5. (A) SAMN@RITC localization within HeLa cells by confocal microscopy. HeLa cells were incubated in the presence of 25 μg mL−1 SAMN@RITC for 24 h and treated with 1 μM calcein acetoxymethyl ester, 15 min before the observation by 3D confocal fluorescence microscopy. Thirty scans on Z axis (250 nm depth) were acquired at 520 nm (calcein AM) and 610 nm (rhodamine). XZ and YZ planes represent the projection on Z axis of vertical sections indicated by the two orthogonal lines on the XY plane. Panel 1: acquisition of signals at 520 and 610 nm. Arrows indicate examples of colocalization of SAMN and Calcein AM; L (lysosome) indicates an acidic vacuolar area in which the signal at 610 nm is present but at 520 nm is absent; I (invagination) indicates an area, external with respect to 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 cells were incubated in the presence of 25 μg mL−1 SAMN@RITC and treated with 1 μM calcein AM, 15 min before the observation by confocal fluorescence microscopy, acquiring fluorescence signal at 520 nm (calcein AM) and 610 nm (rhodamine). Top panels (1): signal at 610 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 24 h incubation.

results to colocalize with green signal of calcein (see arrows in panels 1, 2 and 3), suggesting a possible compartmentalization in nonacidic cellular structures, like pino/endocytotic vacuoles. This inference is supported by the high concentration of SAMN@RITC observed in acidic vacuolar (lysosomal) structures, as shown in reported images (letters L panels 1 and 2), evidencing a strong activity of the vacuolar trafficking, as expected in the case of endocytotic uptake. In the same Figure 5A, a large SAMN@RITC aggregate, located in a plasma membrane invagination outside the cell (indicated by the letter I in panels 1 and 2), can be noted. This observation clearly indicates that large aggregates cannot enter into cells, but that can induce modifications of plasma membrane topology. The 20325



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internalization process results continuous with time, as seen in Figure 5B, where SAMN@RITC are already detectable after two hours incubation and gradually accumulate during the following 22 h. The progressive accumulation of SAMNs within the cells, leading to the formation of macro-aggregates, can be noted. These aggregates are likely located in the lysosomal compartment, 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 incubated for different times (up to 48 h) with increasing concentrations of SAMN@RITC (0 - 200 μg mL−1), and the associated fluorescence was analyzed. The analysis confirms the interaction of cells with SAMN@RITC: the intensity of RITC fluorescence is proportional to nanoparticle concentration and incubation times (see Figure S2 in the Supporting Information). The kinetics of SAMN@RITC fluorescence incorporation by HeLa cells showed a saturation behavior, suggesting that the phenomenon was not due to simple diffusion. However, fluorescence analysis cannot discriminate the contribution of SAMNs simply adsorbed on the outer cell membrane surface or internalized within cell cytoplasm. For this 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 were incubated for 1 h with increasing SAMN@RITC concentrations (0 − 200 μg mL−1) were carried out at 4 °C, temperature at which the almost complete arrest of endocytosis occurs.37 Control experiments were carried out at 37 °C. As depicted in Figure S3, a negligible increase of incorporated SAMN@RITC fluorescence was detected at low temperature, while, at 37 °C, the increase was significant and dosedependent. We cannot a priori exclude the release of RITC from SAMN surface, and it is known that rhodamine accumulates in a membrane potential manner inside the mitochondria.38 In order to exclude this possibility, cell samples were incubated for 30 min with 1 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, which is able to uncouple the mitochondrial membrane potential. No differences in terms of SAMN@RITC fluorescence between HeLa cells populations incubated with or without the uncoupling agent, both at 4 and 37 °C, were observed, indicating that fluorescence inside Hela cells depended only on energy dependent SAMN@RITC incorporation and not on mitochondrial potential, ruling out the presence of free RITC (see Figure S4). Unfortunately, fluorescence cannot be used for the evaluation of the cellular uptake of unlabeled (bare) nanoparticles. For this purpose, an alternative method, consisting in the determination of nanoparticle effect on cellular light scattering properties, was utilized.39 In the case of cellular uptake, the intensity of side scattered light (which provides information on internal structure) increases with the concentration 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 HeLa cells, as function of temperature and bare SAMN concentration. At 4 °C, no appreciable differences of forward and side scattering signals as a function of nanoparticle concentration, were observed, indicating that nanoparticles were not

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

internalized. Conversely, the incubation at 37 °C led to a dramatic increase of side scattering as a function of SAMN concentration, while no relevant modification of forward scattering was detected. At the highest tested concentration (200 μg mL−1) a 600% increase of side scattering was observed at 37 °C, compared to control, while at 4 °C only a 6% increase was 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 superimposable with those observed with bare SAMNs (data not shown). These results clearly show that bare and rhodamine coated SAMNs share the same energy dependent mechanism of cellular uptake, and exclude any role of rhodamine in the internalization process. It was documented that the presence of nanoparticles within cells can induce a detectable damage in terms of reduction of metabolic activity.40 In order to assess if SAMNs are able to produce the same phenomenon, the resazurin reduction assay was carried out on SAMN treated HeLa cells. Resazurin is a vital nonfluorescent dye, which is reduced to the fluorescent resorufin by cellular oxido-reductases, active only in living cells.41 Since this assay constitutes a direct measurement of cell metabolic state, well correlating with living cell density, it can be used for an indirect evaluation of cell viability. Indeed, in our 20326



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Figure 7. Effect of bare and rhodamine coated SAMNs on HeLa cell cycle. Cells were incubated in the presence of increasing concentrations of SAMNs and SAMN@RITC for 24 and 48 h and stained with propidium iodide for cytofluotimetric analysis of the DNA content. Experiments were carried out in triplicate ± SD. In the lower panel, it is reported, for example, the analys of the DNA content concerning Hela cells incubated with bare 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 synthesis phase.

experimental conditions, resazurin reduction rate was found proportional to cell density in a wide range of cellular density (5000−80 000 cells/well). Preliminary experiments on the influence of nanoparticles on the resazurin assay were carried out in culture medium ±10% FBS, without cells or with cellular lysates, and the results altogether excluded interferences in the assay (see Figure S5 in the Supporting Information). The effect of increasing concentrations of bare and rhodamine coated SAMNs (0 - 250 μg mL−1) on Hela metabolic activity was therefore assayed. After 24 h treatment, a significant decrease in resazurine reduction rate (metabolic activity) appeared at nanoparticle concentrations above 100 μg mL−1, being more pronounced with SAMN@RITC (see Figure S5A in the Supporting Information), but no differences were observed below this concentration, indicating negligible nanotoxicological effect of SAMNs.32 After 48 h incubation, the most evident cell damage was found with bare nanoparticles, while cells treated with SAMN@RITC tended to recover the resazurin reduction rate with the prolonging of incubation time (see Figure S5B in the Supporting Information). This different behavior may derive from the different nature of the insult

produced by the two types of nanoparticles, observed only at the highest concentrations. Probably an acute cell damage is exerted by bare SAMNs, while a chronic damage was evident with SAMN@RITC. Finally, the high variability of cell metabolic state, observed at SAMN concentration of 50 μg mL−1, can be interpreted as the threshold SAMN concentration at which HeLa cells are unaffected (see Figure S5B in the Supporting Information). Further information about SAMN effect on HeLa cells was obtained from the determination of the activity of lactate dehydrogenase (LDH), a cytoplasmic enzyme, in the culture medium, due to cellular death and lysis. LDH concentration in the culture medium represents a good indication of cell mortality by necrosis.42 Experiments were performed by exposing cells to increasing concentrations of bare and RITC coated SAMNs (0−250 μg mL−1). The LDH release in the culture medium and LDH amount still present within the cells, were determined at 12, 24, and 48 h. No toxic effects on HeLa cells were observed at SAMN concentrations below 100 μg mL−1, both for SAMNs and SAMN@RITC. SAMNs induced a dose dependent LDH release, which was 21.6% vs control at 20327



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the highest concentration (250 μg/mL), at 12 h incubation (see Figure S6A in the Supporting Information). At longer incubation times, the behavior was confirmed: SAMN toxicity was evident only at the highest concentration (250 μg mL−1), independently of the presence of the rhodamine coating (see Figure S6B in the Supporting Information). Surprisingly, cell mortality, measurable only at 250 μg mL−1 SAMN, was reduced at 48 h incubation. This apparent mortality reduction was probably due to cell replication, which for HeLa cells is approximately 24 h, leading to nanoparticle dilution within cells. Overall, results on cell toxicity rated by LDH release confirmed the data obtained by the determination of resazurin reduction. Nanoparticles, in particular those based on iron oxides, can cause cell damage by induction of oxidative stress.43 The production of reactive oxygen species (ROS) may occurs directly on nanoparticle surface, by spontaneous release of iron ions or by enzymatic nanoparticle degradation within lysosomes.44 Moreover, an increase of ROS production (and of reactive nitrogen species) may derive from the uncontrolled activation of the anti-inflammatory cell response.44 Since ROS production represents an early event, preceding cell damage and death, short incubation times (90 and 180 min) in the presence of SAMNs and SAMN@RITC were chosen to track their possible toxic effects. ROS production by HeLa cells was determined by a specific probe, namely 6-carboxy-2,7′dichlorodihydrofluorescein diacetate (DCFHDA), and flow cytometric analysis.45 We found that bare SAMNs did not stimulate ROS production, at least in the explored concentration range (0−100 μg mL−1). Conversely, a significant ROS production was observed at the highest concentration of SAMN@RITC (100 μg mL−1) after 180 min incubation (see Figure S7 in Supporting Information). It is well-known that cationic rhodamine derivatives can induce mitochondrial damage, both in vivo and in vitro.46 Even if free rhodamine was undetectable within HeLa cells treated with SAMN@RITC under our experimental conditions, we cannot exclude that tiny amount of rhodamine could be released from SAMN surface, interfering with mitochondria metabolism and causing a transient increase of ROS. Proper cell function requires the maintenance of mitochondrial membrane potential, supported by the electron transport chain. Oxidative stress and/or mitochondrial dysfunction are considered to be causes of cellular damage and cell death by apoptosis or necrosis. The mitochondrial membrane potential can be assessed in viable cells by flow cytometry and specific fluorescent probes. In our experiments, tetramethyl-rhodamine (TMR), a lipophilic fluorescent cationic probe, accumulating within the mitochondria as a function of membrane potential, was used. In this case, SAMN@RITC were not tested, due to the superimposition of fluorescent emission by RITC and tetramethyl-rhodamine. Thus, experiments were performed only with bare SAMNs. Incubation of HeLa cells with SAMNs up to 100 μg mL−1, for 90 and 180 min, indicated no effect of nanoparticles on the mitochondrial potential, further demonstrating the safety of SAMNs (see Figure S8 in the Supporting Information). Moreover, it was reported that nanoparticles, depending on their composition, surface coating, size, and so forth, might interfere with the regulation of cell cycle.32 Cell cycle is a highly controlled process, and in each phase consensus checkpoints are present to ensure that the cell is ready to address the crucial step of cell division. The duration of the process normally lasts

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

4. DISCUSSION As described in our previous works, both SAMNs and SAMN@ RITC show an efficient binding capability toward proteins and enzymes and, moreover, the surfaces of these nanoparticles expose friendly environments for protein immobilization, as tested molecules preserved their biological activity after absorption.3,21,22 Generally, bare or functionalized iron oxide nanoparticles reported in literature are able to absorb serum proteins exclusively by electrostatic interactions.33 Conversely, bare SAMNs bind molecules by complexation on undercoordinated iron(III) sites and SAMN@RITC by nucleophilic addition to the isothiocynate moiety. As a consequence, no significant contribution of electrostatic forces in the opsonization 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 iron oxides nanoparticles via electrostatic interactions.33 Bare SAMNS and SAMN@RITC were not able to significantly bind albumin in a complex system, such as fetal bovine serum. Albumin binding on SAMN surface is possible only following incubation in pure BSA solutions under controlled conditions, as demonstrated in the present work, for a long incubation time and in the absence of competitors. The need of a prolonged incubation time may be attributable to low collision efficiency, most likely due to sparse suitable binding sites on nanoparticles surface. Nevertheless, when set in the proper orientation, BSA eventually binds on SAMN surface leading to a monomolecular layer. In this case bovine serum albumin effectively interacts with nanoparticle surface forming a stable monomolecular layer, comprising 6 ± 2 molecules per nanoparticle. Unfortunately, BSA seemed to be denatured by this prolonged incubation, as evidenced by circular dichroism. It should be remembered that other proteins, which effectively bind on SAMNs within few tens of minutes, are not subjected to dramatic alteration of their tridimensional structure upon immobilization, as demonstrated by the maintenance of their biological activity.3,21,22 Within fetal bovine serum (containing 80% BSA), albumin competes with many other proteins for SAMNs and SAMN@ RITC surface binding on iron(III) undercoordinated sites and isothiocynates moieties, respectively, as suggested by Thiele and coauthors on other nanoparticles.48 Interestingly, the two 20328



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when introduced in cell culture medium, spontaneously bind covalently to a small group of specific proteins, producing a self-assembled corona, representing a unique example of magnetic nanoparticle opsonization via covalent binding. Thus, the prolonged nanoparticle uptake indicates a correlation between internalization and stability of covalently bound protein corona. We suggest that SAMN based nanobiocomposites can be employed for the preparation of self-assembled opsonized nanoparticles as future candidates for diagnostic and therapeutic applications in biomedicine.

different surface chemistries led to the formation of the same protein shell, mainly constituted by different proteins, such as APO-B100, fetal hemoglobin α and β subunits, trombospondin1 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 promoting nanoparticles uptake by cells.49 The present study on Hela cells evidenced that the proposed nanoparticles (SAMNs and SAMN@RITC) show the same behavior in terms of internalization efficiency and cytotoxicity, suggesting that they are similarly recognized by cells, indeed they resulted coated predominantly by the same proteins. Among the different proteins involved in the binding with nanoparticle surface, as evidenced by gel electrophoresis and mass spectrometry results, α-2-HS-glycoprotein was the most represented and APO-B100 the most tightly bound. These proteins could play an important role in nanoparticle internalization in cells,50 as already evidenced by Thiele and coauthors.48 They also reported that serum protein binding on nanoparticle surface (opsonization) is lost after 2 h incubation. In the present case, SAMNs and SAMN@RITC coated with serum proteins, mainly with α-2-HS-glycoprotein, can be internalized even after 24 h incubation, confirming a correlation between binding efficiency and stability of protein-nanoparticle complexes. Only proteins presenting correctly positioned anchoring groups on their surface can quickly and efficiently adsorb on SAMNs and SAMN@RITC. For these kinds of proteins, the nanoparticle surface represents a friendly environment, leading to a stable protein shell, easily recognizable by cells for a long time. Moreover, conversely to recent findings,51,52 in which the spontaneous formation of a protein corona led to a loss of targeting capabilities of nanoparticles, SAMNs and SAMN@RITC are available for projecting a synthetic, functional, and stable protein corona, thus avoiding further uncontrolled serum protein binding. This approach can be exploited for the preparation of properly targeted magnetic nanoparticles for different cell lines, overcoming the role of culture media on spontaneous protein absorption on nanoparticle surface, as evidenced by Laurent and co-workers,53 and allowing the selective recognition by different cell lines. This cell recognition property was recently defined as “cell vision”,54 emphasizing that what the cell “sees”, when it is faced with nanoparticles, is most likely dependent on the cell type. The present report evidences the importance of studying protein adsorption on nanoparticle surfaces in complex protein mixtures for further biomedical applications. In the present case, protein absorption experiments, on two maghemite nanoparticle formulations, carried out in fetal fetal bovine serum led to the preparation of iron oxide nanoparticles characterized by a suitable and stable protein coating for cell internalization. Moreover, this study allows one to gather informative data on SAMN internalization and cytotoxicity and gives important indications for future preparations of selectively opsonized nanoparticles. SAMN based nano-biocomposites, due to their stability and ability to stimulate cell uptake, are possible future candidates for a wide range of biomedical applications, from drug delivery, cancer therapy, and theranostics.

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ASSOCIATED CONTENT

S Supporting Information *

Intrumentation and methods, comprising synthesis of iron oxide nanoparticles (SAMN), preparation of rhodamine bound nanoparticles (SAMN@RITC), immobilization of bovine serum albumin on SAMNs, opsonization of SAMNs by fetal bovine serum (FBS), detachment of FBS proteins bound to SAMNs, SDS-PAGE electrophoresis, in gel protein digestion, protein identification by mass spectrometry, cell culture, confocal microscopy imaging, cytofluorimetric analysis, determination of lactate dehydrogenase activity, resazurin fluorimetric assay, cytofluorimetric analysis of mitochondrial activity and reactive oxygen species production (ROS) and cell cycle analysis. Figures regarding the optical spectrum of SAMN suspension, the SAMN interaction with HeLa cells determined by flow cytometry, SAMN@RITC uptake by HeLa cells determined by flow cytometric analysis of fluorescence, SAMN@RITC uptake by HeLa cells determined by flow cytometry analysis of fluorescence, metabolic activity of HeLa cells following SAMN treatment, HeLa cell viability determined by LDH activity measurements in the culture medium, reactive oxygen species production by bare and RITC coated SAMNs on HeLa cells, and effect of SAMNs on mitochondrial potential of HeLa cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0039-049-8276863. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present experimental work was partially funded by an Italian Ministry grant (60A06-0587/12). The authors gratefully acknowledge the support by the Operational Program Research and Development for Innovations - European Regional Development Fund (project CZ.1.05/2.1.00/03.0058) and by the Operational Program Education for Competitiveness European Social Fund (project CZ.1.07/2.3.00/20.0155) of the Ministry of Education, Youth and Sports of the Czech Republic. The Foundation for Advanced Biomedical Research and VIMM are grateful to the “Veneto Banca” Holding for funding the acquisition of the MALDI-TOF/TOF mass spectrometer. The University of Padova is grateful to the “Cassa di Risparmio di Padova e Rovigo” Holding for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer.

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5. CONCLUSIONS In the present case, our nanoparticles form stable colloidal suspensions without any preliminary polymeric coating and,

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