A plasma protein corona enhances the biocompatibility of Au ... - Core

Biomaterials 68 (2015) 77e88

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A plasma protein corona enhances the biocompatibility of Au@Fe3O4 Janus particles Lisa Landgraf a, Carolin Christner a, Wiebke Storck b, Isabel Schick c, Ines Krumbein d, €ber a, €hring a, Katja Haedicke a, Karl Heinz-Herrmann d, Ulf Teichgra Heidi Da d c b, ** , Ingrid Hilger a, * Jürgen R. Reichenbach , Wolfgang Tremel , Stefan Tenzer a

Institute of Diagnostic and Interventional Radiology, Division of Experimental Radiology, University Hospital Jena, Erlanger Allee 101, 07747 Jena, Germany Institute for Immunology, University Medical Center of Mainz, Langenbeckstraße 1, D-55101 Mainz, Germany Institute for Inorganic Chemistry and Analytical Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, D-55128 Mainz, Germany d Medical Physics Group, Institute of Diagnostic and Interventional Radiology, Jena University Hospital, Friedrich Schiller University Jena, Philosophenweg 3, D-07743 Jena, Germany b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 March 2015 Received in revised form 24 July 2015 Accepted 31 July 2015 Available online 1 August 2015

Au@Fe3O4 Janus particles (JPs) are heteroparticles with discrete domains defined by different materials. Their tunable composition and morphology confer multimodal and versatile capabilities for use as contrast agents and drug carriers in future medicine. Au@Fe3O4 JPs have colloidal properties and surface characteristics leading to interactions with proteins in biological fluids. The resulting protein adsorption layer (“protein corona”) critically affects their interaction with living matter. Although Au@Fe3O4 JPs displayed good biocompatibility in a standardized in vitro situation, an in-depth characterization of the protein corona is of prime importance to unravel underlying mechanisms affecting their pathophysiology and biodistribution in vitro and in vivo. Here, we comparatively analyzed the human plasma corona of Au-thiol@Fe3O4-SiO2-PEG JPs (NH2-functionalized and non-functionalized) and spherical magnetite (Fe3O4-SiO2-PEG) particles and investigated its effects on colloidal stability, biocompatibility and cellular uptake. Label-free quantitative proteomic analyses revealed that complex coronas including almost 180 different proteins were formed within only one minute. Remarkably, in contrast to spherical magnetite particles with surface NH2 groups, the Janus structure prevented aggregation and the adhesion of opsonins. This resulted in an enhanced biocompatibility of corona sheathed JPs compared to spherical magnetite particles and corona-free JPs. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Janus particles Protein corona Nanotoxicity Nanomedicine In vivo imaging

1. Introduction Bioimaging techniques, including ultrasound, computed X-ray tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), are essential tools not only for scientific research, but also for clinical diagnostics. The synergistic combination of multiple comprehensive imaging techniques in diagnostics enables a faster, more accurate, and physically less demanding prognosis [1]. These latest developments have increased scientific interest and clinical need for multimodal

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S. Tenzer), [email protected]. de (I. Hilger).

contrast agents, which could enhance safety and reduce adverse effects by limiting the amount and dose of different contrast agents, improve patient care, and, at the same time, lower costs. Au nanoparticles display extraordinary optical properties relying on surface plasmon resonance (SPR), enabling their widespread use and high potential for biomedical applications, including simultaneous cell-imaging and photothermal therapy [2e4], multi-photon microscopy [5], and colorimetric sensing of adsorbed biomolecules [6,7]. Moreover, they are emerging as next generation contrast agents for computed X-ray tomography (CT) [8,9]. Similarly, magnetic nanoparticles such as magnetite (Fe3O4) constitute a class of nanomaterials that has attracted much interest for biomedical applications [10,11] including therapeutics for hyperthermia, drug delivery, and diagnostics [12e14]. Au@Fe3O4 JPs are multifunctional heteroparticles harboring two

http://dx.doi.org/10.1016/j.biomaterials.2015.07.049 0142-9612/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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L. Landgraf et al. / Biomaterials 68 (2015) 77e88

discrete domains formed by different materials. Their tunable composition and morphology confer multimodal and versatile capabilities for use as contrast agents and drug carriers in future medicine, whose potential applications in different fields is only emerging [15e19]. The intrinsic appeal of JPs is their tunable and controllable asymmetric structure, providing spatially controlled functionalities down to the nanoscale. Especially biomedical applications profit from the synergistic potential for multiplexing, multi-level targeting, and combination therapies. The availability of two different surfaces for multiple drug and target loading without steric hindrance from other components is of particular importance. In addition, the two different nanoparticle domains can be used for different imaging modalities, e.g. gold for X-ray based imaging [20,21] and magnetite for magnetic resonance imaging (MRI) [22e24]. The metallic nature of the cores offers versatile combinations of minimal invasive therapy approaches, like photothermal ablation by exploiting the absorbance of light by the gold and magnetic hyperthermia by utilizing the superparamagnetic behavior of iron oxide [12,25e28]. A major downside of most inorganic nanoparticles is their colloidal instability due to agglomeration based on mutual attraction by van der Waals forces. As most biomedical applications require administration of nanoparticles into the body via intravenous injection, an essential prerequisite for the implementation of bionanotechnological applications is to ensure sufficient colloidal stability under physiological conditions, i.e. the in vitro and in vivo biocompatibility is an essential barrier for an application of new nanomaterials. After application, the nanoparticles will typically be transported via the blood system and will react with blood proteins and the immune system [29e31]. The adhesion of proteins to the nanoparticles in a specific biological environment determines their fate in the body. In particular, immune cells can be activated which provides the clearance of the particles by the mononuclear phagocyte system [32e34]. Even though nanoparticle protein coronas have been intensively investigated for liposomes or symmetric and isotropic polystyrene, magnetite, and silica nanoparticles [30,35e37], it is yet unclear whether asymmetric and amphiphilic Au@Fe3O4 nanoparticles exhibit similar features regarding the formation of a protein corona similar to spherical nanoparticles [30,38e40]. In view of the widespread biomedical applications of JPs, they have a similar biodistribution [15] as their spherical and isotropic component constituent blocks. Here, we applied a comprehensive experimental approach to study the formation of a protein corona of Au@Fe3O4 Janus nanoparticles and its effects on the in vitro and in vivo situation. To unravel the effect of the asymmetric nanoparticle morphology on the adhesion of plasma proteins, we compared JPs with spherical Fe3O4 nanoparticles of comparable size and composition. Both particle types contain a silica shell at the Fe3O4 domain modified with a short-chained PEG, while the gold part of the JPs was coated with thiol groups (Fe3O4-SiO2-PEG vs. Au-thiol@Fe3O4-SiO2-PEG). Since surface NH2 groups are typically used for further functionalization of nanoparticles, the magnetite domain of the JPs and the symmetric Fe3O4 particles were amine functionalized (Fe3O4-SiO2PEG-NH2 vs. Au-thiol@Fe3O4-SiO2-PEG-NH2) to gain information on the impact of surface functionalization and nanoparticle morphology on the composition of the protein corona. We applied a well-established quantitative proteomic workflow [30,41] for the detailed characterization of the protein corona. Additionally, changes in the nanoparticle stability and surface properties were analyzed by size and zeta potential measurements after incubation in human plasma. To gain further insight into the pathophysiological effects on cellular metabolism, we compared cellular ATP

levels and intracellular uptake of nanoparticles with a corona and particles with no corona using human endothelial cells, which represent the first barrier after intravenous (i. v.) injection. Lastly, the in vivo biodistribution of JPs and their MRI imaging properties as well as the organ iron content was determined after i.v. application, revealing general feasibility of Janus particles as blood pool contrast agent. 2. Materials and methods 2.1. Synthesis of asymmetric and spherical nanoparticles The Au@Fe3O4 and spherical Fe3O4 nanoparticles were synthesized as described previously with thiol groups at the gold domain and a silica-PEG shell at the iron oxide domain (Au-thiol@Fe3O4SiO2-PEG and Fe3O4-SiO2-PEG) [15,16]. Gold nanoparticles were prepared by a modified procedure [42] followed by the synthesis of Au@Fe3O4 heteroparticles using a seed-mediated chemical protocol [43,44]. The heating of 15 mg of Au nanoparticles, 6 mmol (2 mL) of oleylamine, 6 mmol (2 mL) oleic acid, and 20 mL of 1-octadecene in an argon atmosphere up to 120 C was followed by the injection of 2 mmol (0.27 mL) of iron pentacarbonyl. Particle collecting was carried out after cooling down to room temperature by adding isopropanol (five times the volume of the mixture), a centrifugation step (9344 g, 10 min) and re-dispersion in hexane. Monodisperse isotropic iron oxide nanoparticles were synthesized by thermal decomposition of the iron(III)oleate [45,46] followed by the coating with a silica-PEG [47]. For comparison of functionalization, NH2groups were attached via (3-aminopropyl)-triethoxysilane (APS) to the iron oxide core. 2.2. Particle characterization Core sizes, hydrodynamic diameters and charge were determined by transmission electron microscopy (TEM) (Philips EM 420, LaB6 cathode, 120 kV acceleration voltage) of 150 individual nanoparticles, dynamic light scattering (DLS), and zeta potential (z) measurements (Zeta sizer, Malvern), respectively. For DLS and zeta potential determinations 50 mg/mL Fe(II)/(III) of nanoparticles were incubated in water or in human plasma for 1 h at 4 C. All nanoparticle suspensions were centrifuged through a 0.7 M sucrose cushion at 18.000 g for 25 min to ensure the same conditions throughout all experiments [30]. The nanoparticle pellets were suspended in water and transferred into measurement cuvettes (Malvern). The average hydrodynamic diameter was determined from 5 measurements. Zeta potential measurements were performed in triplicate, each with 10 runs at 25 C. 2.3. Human blood and plasma preparation Blood from six different healthy volunteers was transferred to smonovettes (citrate coated tubes) (Sarstedt). For plasma preparation 50 mL blood was collected in a falcon tube (Eppendorf). The tubes were centrifuged for 12 min at 4000 g to pellet the blood cells [41]. The plasma supernatant was aliquoted and stored at 80 C. For experiments, plasma was thawed and centrifuged for 2 min at 12,000 g/4 C to pellet protein precipitates. All experiments were performed according to the requirements of the local ethics committee. 2.4. Incubation of nanoparticles with human plasma Nanoparticles (100 mg/mL Fe(II)/(II)) of the different formulations were incubated in 500 mL of plasma for 1 min or 1 h at 4 C. To pellet the protein-nanoparticle complexes, the nanoparticle plasma


mixtures were loaded on 700 mL of 0.7 M sucrose (Roth) and centrifuged for 25 min at 18,000 g/4 C [30]. The resulting supernatants were used for quantitative protein analysis. The nanoparticle pellets were resuspended in wash buffer (103.5 mM NaCl, 5.3 mM KCl, 5.6 mM Na2HPO4, 1.4 mM KH2PO4, 23.8 mM NaHCO3, pH 7.4) [41]. Afterward, NP-pellets were washed with buffer at least three times by repeated centrifugation; all supernatants were collected. To elute the proteins from the nanoparticles for SDS-PAGE, the pellets were resuspended in SDS-sample buffer (62.5 mM TriseHCl pH 6.8; 2% w/v SDS, 10% glycerol, 50 mM DTT, 5% 2mercaptoethanol, 0.01% w/v bromphenol blue) and boiled for 15 min at 95 C in a water bath. 2.5. Quantitative protein analysis using Bradford For quantitative analysis of proteins bound to the nanoparticles, supernatants after nanoparticle centrifugation through the sucrose cushion were processed for a colorimetric Bradford method [48]. Analysis was performed in triplicate. 2.6. SDS-PAGE For the SDS-PAGE we used the eluted and boiled protein samples (see above). A SDS-polyacrylamide gel electrophoresis (PAGE) (1D) with all samples was carried out using a 12% polyacrylamide gel [30]. Visualization of the proteins was performed via silver nitrate staining as described previously [49].

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Fibrinopeptide B (100 fmol/mL), was delivered by the auxiliary pump of the LC system at 500 nL/min to the reference sprayer of the NanoLockSpray source of the mass spectrometer. Mass spectrometric analysis of tryptic peptides was performed using a Synapt G2-S mass spectrometer (Waters Corporation, Manchester, UK) operated in data-independent acquisition mode as described in Ref. [51]. The spectral acquisition time in each mode was 0.6 s with a 0.05-s interscan delay. In low energy MS mode, data were collected at constant collision energy of 4 eV. In elevated energy MS mode, the collision energy was ramped from 16 to 40 eV during each 0.6-s integration. One cycle of low and elevated energy data was acquired every 1.3 s. The radiofrequency (RF) amplitude applied to the quadrupole mass analyzer was adjusted such that ions from m/z 350 to 2000 were efficiently transmitted, ensuring that any ions observed in the LC-MS data less than m/z 350 were known to arise from dissociations in the collision cell. All samples were analyzed in triplicate. Obtained raw data were processed with ProteinLynxGlobalServer v.3.0.1 and obtained spectra were searching against the human UniProtKB/Swiss prot reference proteome database (UniProtKBrelease 2012_07, 20,231 entries). Sequence information of enolase 1 (S. cerevisiae) was added to the data bases to conduct absolute quantification (5) using the ISO Quant software [50]. The experimental data were typically searched with a 2-ppm precursor and 6-ppm production tolerance, respectively, with one missed cleavage allowed and fixed carbamidomethylcysteine and variable methionine oxidation set as the modifications. 2.8. Cell viability and nanoparticle uptake

2.7. Label-free quantitative proteomic analysis of corona components After isolation of the nanoparticle protein complexes through the sucrose cushion and washing the pellets in wash buffer (103.5 mM NaCl, 5.3 mM KCl, 5.6 mM Na2HPO4, 1.4 mM KH2PO4, 23.8 mM NaHCO3, pH 7.4), they were suspended in 7 M urea, 2 M thiourea and 2% of CHAPS (all components purchased from Roth). Eluted corona proteins (20 mg) were subjected to tryptic digestion using a modified filter aided sample preparation protocol as described in Ref. [50]. Briefly, eluted corona proteins were loaded on the filter, and detergents were removed by washing three times with 8 M urea buffer. The proteins were reduced using DTT, alkylated using iodoacetamide, and the excess reagent was quenched additional DTT and washed through the filters. Buffer was exchanged to 50 mM NH4HCO3 and proteins digested overnight by trypsin (Trypsin Gold, Promega, enzyme to protein ratio of 1:50). Peptides were recovered after overnight digestion by centrifugation and two additional washes using 50 mM NH4HCO3. Flow throughs were combined, lyophilized and redissolved in 20 ml 0.1% formic acid by sonification. Tryptic peptide solutions were diluted with aqueous 0.1% v/v formic acid to a concentration of 200 ng/ml and spiked with 25 fmol/ul of enolase 1 (Saccharomyces cerevisiae) tryptic digest standard (Waters Corporation). Nanoscale UPLC separation of tryptic peptides was performed with a nano Acquity system (Waters Corporation) equipped with a CSH-C18 1.8 mm, 100 mm 250 mm analytical reversed-phase column (Waters Corporation) in direct injection mode as described before [50]. 300 ng of total protein was injected per technical replicate. Mobile phase A was water containing 0.1% v/v formic acid, while mobile phase B was ACN containing 0.1% v/v formic acid. Peptides were separated with a gradient of 5e40% mobile phase B over 34 min at a flow rate of 300 nL/min, followed by a 4-min column rinse with 90% of mobile phase B at 500 nl/min. The columns were re-equilibrated at initial conditions for 6 min. The analytical column temperature was maintained at 45 C. The lock mass compound, [Glu1]-

Human micro vascular endothelial cells (HMEC-1; Centers for Disease Control and Prevention) were cultured in Gibco™ MCDB 131 medium supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX™ I (100; Life Technologies GmbH), 1 mg/mL hydrocholesterol (Sigma Aldrich Chemie GmbH), 10 ng/mL epidermal growth factor (Life Technologies GmbH). To determine the effects on cell metabolism an ATPLite (PerkinElmer) assay was used. Cells were plated onto a plastic matrix at a density of 1.2 104 cells/cm2 then allowed to grow for 24 h prior to nanoparticle exposure. Nanoparticle concentrations ranging from 5 to 50 mg/mL Fe(II)/(III) were preincubated in water or in human plasma for 1 h at 4 C as described above. All pelleted nanoparticles through the sucrose were suspended in complete culture medium and transferred to the cells and incubated for 24 h at 37 C in a 5% CO2 humidified environment. After several washing steps, the cellular ATP level was assessed according to the manufacturer's instructions. The luminescent signals emerging from the transformation of D-luciferin to oxyluciferin were measured using the LUMIstar Galaxy (BMG LABATECH GmbH) system. The determined ATP levels after nanoparticle exposure were normalized to non-treated control cells. The internalization of nanoparticles was determined after washing the cells during the ATP assay by microscopy (EVOS, PEQLAB GMBH). 2.9. In vivo biodistribution in mice During all experiments, animals were anesthetized with 2.5% isoflurane (Aktavis). All experiments were in accordance with international guidelines on the ethical use of animals and were approved by the regional animal care committee (reg. number 02069/11). At day one 5.4e6.4 mg Fe(II/III) per gram body weight of the different nanoparticle formulations were injected into female athymic nude mice (Hsd:Athymic Nude-Foxn1nu) (Harlan Laboratories) intravenously. After 24 h, MRI measurements were performed on a clinical 3T whole-body system (TIM Trio, Siemens

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Healthcare) using a dedicated 8-channel whole-body mouse coil [52] (Rapid Biomedical). During the MRI scan the animals were monitored [53]. The used imaging sequence was a T2* -sensitive spoiled gradient echo sequence with TE/TR/a ¼ 4.91 ms/16 ms/22 , a resolution of 0.31 0.31 0.25 mm3 and, with 2 averages, a total acquisition time of 8 min 46 s. To measure the iron content of the organs all mice were sacrificed for organ preparation after additional 24 h of incubation. 2.10. Measurements of organs iron content Iron content of organs was determined using flame atomic absorption spectrometry. The organs of three mice of each group were isolated, each organ was cut into three slices, dried for 48 h at 37 C and transferred to quartz glass tubes. During this transfer process, the weight of the samples was determined. Incineration of the dried organ slices was performed in a solution consisting of 3 fractions of 65% nitric acid (HNO3) and 2 fractions of 70% perchloric acid (HClO4) at 250 C. The samples were dissolved in HNO3, after mixing they were diluted in n/10 hydrogen chloride (HCl) (Roth) directly in the analysis vials (Analytik Jena). Afterward, analysis of the iron content was done using an AAS5 FL spectrometer (Analytik Jena). All measurements were performed in triplicates. Standard solutions (5e50 mmol Fe l 1 in 0.1 N hydrochloric acid; Merck) were for calibration. The determined iron concentrations were used for the mathematical correction of the amount of iron per gram of the corresponding dried organ. 2.11. Statistical analysis The data were analyzed using a paired Student's t-test. Statistical significance was calculated by comparing the different experimental samples. P values < 0.05 are considered as significant. 3. Results 3.1. The protein corona stabilizes Janus particles in suspension Electron microscopy of the nanoparticle formulations (Fig. 1A) revealed core sizes for the gold domain of 6.1 ± 0.3 nm and for the iron oxide core of 16.1 ± 1.4 nm. The centrifugation process through a sucrose cushion to isolate the protein-nanoparticle complexes enhanced the aggregation of the nanoparticles in water. Interestingly, the presence of a protein corona after nanoparticle incubation in human plasma stabilized all nanoparticle formulations, preventing aggregation. This stabilization was indicated by a reduced hydrodynamic diameter (Fig. 1B). Notably the NH2-functionalization of the spherical Fe3O4-SiO2-PEG-NH2 nanoparticles resulted in an assembly of pristine (i.e. not containing a protein corona) nanoparticles into large agglomerates, which was not observed after incubation with plasma, indicating that the presence of protein corona prevents nanoparticle agglomeration. All nanoparticle variants displayed a positively charged surface in water, which was further enhanced by NH2-functionalization, for Au-thiol@Fe3O4-SiO2-PEG from 5.3 to 17.9 mV and for the Fe3O4-SiO2PEGfrom 3.6e22.9 mV, likely due to a higher protonation level caused by the NH2 groups (Fig. 1C). The NH2-functionalization of the spherical variants increased the charge to a higher extent than that of the JPs (Fig. 1C). Importantly, the zeta potentials became negative after incubation in human plasma for all investigated nanoparticles indicating the attachment of oppositely charged plasma proteins (Fig. 1C). The amphiphilic Janus character reduced the agglomeration tendency caused by NH2 groups, but generally the particle morphology had no impact on stability and charge.

3.2. NH2-functionalization modifies the protein corona composition The characterization of proteins constituting the protein corona of Au-thiol@Fe3O4-SiO2-PEG nanoparticles provides essential data concerning nanoparticle protein interactions due to the asymmetric Janus structure. Analysis of corona components by SDSPAGE revealed relatively low protein binding for all nanoparticles, which might be caused by the PEG surface functionalization (Fig. 2A). In order to characterize the effect of incubation time on the protein corona assembled on the surface of the particles, we analyzed the corona composition after one (10 ) and sixty minutes (600 ) of incubation with plasma. Corona formation occurred rapidly already within a minute for all nanoparticle formulations, indicating the presence of major protein components with molecular masses of 180, 100, 70, 45, and 20 kDa (Fig. 2A). The intense band around 70 kDa represents serum albumin. There was no significant difference in protein adsorption between Au-thiol@Fe3O4-SiO2-PEG and spherical Fe3O4-SiO2-PEG nanoparticles. Notably, we observed additional bands above 100 and 180 kDa for NH2-functionalized nanoparticles after sixty minutes of incubation (Fe3O4-SiO2-PEGNH2 and Au-thiol@Fe3O4-SiO2-PEG-NH2) (Fig. 2A). Since these additional bands were not detectable for non-amine functionalized JPs, we conclude that the association is NH2-functionalization dependent (Fig. 2A). The kinetics of plasma protein/nanoparticle binding showed an increase in protein adsorption with time for JPs as compared to spherical nanoparticles irrespective of the presence of NH2 groups (Fig. 2C). In summary, Au-thiol@Fe3O4-SiO2-PEG JPs displayed a higher tendency to form a protein corona than spherical Fe3O4-SiO2-PEG nanoparticles. 3.3. Janus character reduces adverse effects of NH2functionalization To identify biologically relevant plasma proteins adsorbed to the different nanoparticle formulations, we characterized the protein corona using label-free quantitative liquid chromatography mass spectrometry (LC-MS) [30]. Generally, serum albumin was identified as the most abundant corona protein (Table 1). Surprisingly, the NH2 groups at the Fe3O4 domain of the JP had a smaller impact than the NH2 groups on spherical Fe3O4 particles (Au-thiol@Fe3O4SiO2-PEG vs. Fe3O4-SiO2-PEG-NH2) (Table 1). Histidine-rich glycoprotein was detected as the most abundant protein on NH2-functionalized Fe3O4 particles (Table 1). Classifying the corona proteins according to their molecular mass and isoelectric point revealed that proteins with sizes between 30 and 100 kDa constitute the majority of all corona proteins (Table 1). The most abundant protein on Au-thiol@Fe3O4-SiO2-PEG, Au-thiol@Fe3O4-SiO2-PEG-NH2, and Fe3O4-SiO2-PEG particles was serum albumin with a negative net charge (IEP 5.86) indicating that nanoparticles with a zeta potential between þ3 and þ20 mV preferentially attract negatively charged proteins. In contrast, spherical Fe3O4-SiO2-PEG-NH2 particles displayed a zeta potential higher than þ20 mV which could explain the binding of the neutral histidine-rich glycoprotein (Table 1). Next, we performed a bioinformatic classification of the corona proteins categorized in groups according to their biological activities in the blood system (Fig. 3). Acute phase proteins like ceruloplasmin, haptoglobin, and serum amyloid are known as proteins in inflammatory response [54,55]. Furthermore, proteins of the complement system and coagulation proteins play an important role in the human immune response [56,57]. Overall, Fe3O4-NH2 particles adsorbed less lipoproteins (compared to all other NPs), acute phase proteins, and less proteins classified as “other” (including serum albumin) (Fig. 3). In contrast, they adsorbed a major portion of the coagulation protein “histidine-rich glycoprotein” and more proteins of the complement system compared to


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Fig. 1. Schematic and microscopical depiction and the physicochemical characterization of the asymmetric Au@Fe3O4 and the spherical control nanoparticles. (A) Schematic illustration of the used nanoparticle formulations with a thiole functionalized 3.5 nm sized gold core and silica-PEG coated 16 nm sized iron oxide core with additional NH2 groups as functionalization. (B) TEM images of the nanoparticle formulations. Scale bar: 50 nm (C) Hydrodynamic diameters and (D) zeta potentials of the nanoparticle variants determined after incubation in water or human plasma. PC: protein corona. *p < 0.05, **p < 0.005.

the other nanoparticles variants (Fig. 3). JPs (both NH2-functionalized and non NH2-functionalized) showed, independent of the amine-groups, similar tendencies with a higher amount of proteins from the complement system. Additionally, the corona constitution did not display major changes compared to the Fe3O4-SiO2-PEGNH2-nanoparticles.

3.4. The protein corona specifically enhances the biocompatibility of JPs Using human endothelial cells as a model system reflecting the blood situation, we demonstrated the pathobiological relevance of the corona. Cells incubated with corona-coated Au-thiol@Fe3O4SiO2-PEG nanoparticles showed increased cellular ATP levels compared to cells treated with pristine particles (Fig. 4A, upper left panel). This effect was independent of the presence of amino groups (Fig. 4A, right panel). In contrast, spherical Fe3O4-SiO2-PEG nanoparticles associated to a corona reduced the metabolic cell activity (Fig. 4A, lower left panel). Interestingly, spherical nanoparticles exhibited a better biocompatibility without the protein corona. Next, we monitored the effect of corona assembly on the cellular nanoparticle uptake by optical microscopy. Our analysis revealed an enhanced uptake of nanoparticles with coronas by HMEC-1 cells, which was detectable for all nanoparticle variants irrespective of the NH2-functionalization (Fig. 4B, right panels).

3.5. Janus particles enable generation of T2* -contrast in vivo To analyze distribution of JPs in vivo, they were injected intravenously via the tail vein (Fig. 5). Magnetic resonance imaging demonstrated that Au-thiol@Fe3O4-SiO2-PEG JPs can be used for in vivo imaging due to the generation of T2* -contrast similar to that of the spherical Fe3O4-SiO2-PEG particles (Fig. 6A). Importantly, mice that had been i.v. injected with JPs showed no cytotoxic or thrombotic symptoms. Since PEGylation helped to prolong the blood circulation time of nanoparticles [58] we also used PEG at the Fe3O4 domains (Fe3O4-SiO2-PEG and Au-thiol@Fe3O4-SiO2-PEG). MRI showed an accumulation of the Janus and spherical nanoparticles in the liver and spleen 24 h post injection (Fig. 6A), the typical organs of the lymphatic and phagocytic active system. The T2* -sensitive images demonstrated a strong intensity difference of these organs between treated and native mice (Fig. 6A). Determination of organ iron content flame atomic absorption spectroscopy indicated that only half of the applied JPs were taken up by cells of the monocyte phagocyte system and transported into the liver or were directly internalized by liver cells 48 h post injection (Fig. 6B). Iron content of the liver was increased from 0.89 mg Fe/g tissue for native mice up to 1.18 mg Fe/g tissue for the JP-treated and up to 1.11 mg Fe/g tissue for spherical nanoparticletreated mice (Fig. 6B). Notably, only approximately 58% of the injected 136 mg Fe was transferred to the liver indicating that the remaining particles probably still strayed through the body or were taken up by other cells.

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Fig. 2. NH2-functionalization modifies corona composition. (A) SDS-PAGE stained with silver nitrate visualizes the bound proteins to the different nanoparticle formulations. Molecular weight and incubation time is indicated. (B) SDS-PAGE visualizes whole plasma proteins. (C) Amount of bound proteins was determined using standard Bradford technique. *p < 0.05.

4. Discussion In the nanomedical field, Janus particles provide a high potential for the development strategies for tumor detection and therapy [19,59,60]. However, it is unclear whether their special amphiphilic properties and their asymmetric morphology impact their stability in physiological environments. Upon contact with biological fluids, nanoparticles bind proteins, establishing a protein corona in as little as 30 s [30]. Recently, several studies investigated the formation of the protein corona on different types of nanoparticles [30,61,62], including silica and polymer nanoparticles [30,38,63] as well as gold and silver nanoparticles [62]. In addition to the nanoparticle material, protein adsorption depends on nanoparticle size and charge [41,63]. As the protein corona constitutes the “bio/ nano interface”, which affects the biological identity of the nanoparticles by determining their biocompatibility and transport [29,30] we have characterized the formation of a protein corona on Au-thiol@Fe3O4-SiO2-PEG JPs in detail using a quantitative proteomics approach and linked our corona data to effects on cells in vitro and in vivo. For our experiments, we selected Au-thiol@Fe3O4-SiO2-PEG JPs with and without amino functionalization, as NH2 groups are frequently used to link cargo ligands like drugs, proteins, DNA and antibodies to nanoparticle surfaces [64e66] and compared them to the respective spherical Fe3O4-SiO2-PEG control particles. Stability measurements of the hydrodynamic diameter and the zeta potentials revealed similar results for both JPs and spherical control particles. Size and charge of JPs did not reveal major deviations from those of the spherical formulations, i.e. the amphiphilic structure did not markedly affect the general physicochemical characteristics of JPs. Interestingly, while NH2functionalized spherical Fe3O4-SiO2-PEG-NH2 nanoparticles

dispersed poorly in water, the Janus structure prevented agglomeration of Au-thiol@Fe3O4-SiO2-PEG-NH2 particles, thereby stabilizing the particle in solution. A tendency to agglomeration caused by surface NH2-groups has been described previously [15,67], showing an increase of hydrodynamic diameters of aminemodified polystyrene nanoparticles as compared to control nanoparticles [67]. As only the Fe3O4-SiO2-PEG-NH2 particles showed a zeta potential >þ20 mV in water, we hypothesize that particle aggregation is favored by charge effects. In the NH2-PEG functionalized spherical Fe3O4 nanoparticles the terminal NH2 group (pK z 9) will be protonated in buffered solution, i.e. the zeta potential becomes more positive, and therefore a large hydration shell will develop around these spherical articles which leads to the observed increase of the hydrodynamic radius. In contrast, in the Au@Fe3O4 JPs only the iron oxide domain, functionalized with NH2terminated PEG is strongly involved in the interaction with the solution, whereas the unfunctionalized Au domain is hydrophobic and remains inert. This steric “protection” by the Au domain blocks polar interaction with the solution and prevents a significant increase of the hydrodynamic radius. The smaller amount of surface ligands (compared to the spherical particles) also prevents protonation of the terminal NH2 groups and a corresponding change of the zeta potential. Upon contact with serum proteins, all particles displayed a negative surface charge. This may prevent inter-nanoparticle interactions and thus aggregation via inter-particle van der Waal's forces [68,69], thereby reducing nanoparticle agglomeration. An in-depth characterization of adsorbed corona proteins is essential to investigate corona effects on the biological identity of JPs and their interactions with human cells. Towards this purpose, we provide an overview of bound plasma proteins by 1D-SDSPAGE. Next, using an established label-free quantitative proteomics

Table 1 Top 20 of the most-abundant corona proteins (ppm) detectable after 60 min of plasma exposure with their isoelectric point (IEP) and molecular weight (MW). No.

Au@Fe3O4

1

IEP Au@Fe3O4-NH2

MW [kDa]

Serum albumin

71

5.86 Serum albumin

71

5.86 Serum albumin

71

2

Fibrinogen alphachain

96

5.61 Fibrinogen alphachain

96

3

Fibrinogen betachain

57

37

4

Ig gamma-1 chain C region

37

8.25 Ig gamma-1 chain C region 8.18 Fibrinogen betachain

5.61 Ig gamma-1 chain C region 8.18 Fibrinogen alphachain

57

5

Fibrinogen gammachain

52

5.24 Fibrinogen gammachain

52

6

Apolipoprotein(a)

515

5.5

49

7 8 9

Apolipoprotein A-I Apolipoprotein B-100 Serotransferrin

31 516 79

5.43 Apolipoprotein(a) 515 6.58 Apolipoprotein A-I 31 6.75 Ig kappa chain C region 11

10 11 12

Complement C3 Ig kappa chain C region Haptoglobin

188 11 45

5.96 Apolipoprotein B-100 5.5 Complement C3 6.12 Clusterin

13

102

4.9

Serotransferrin

14

Band 3 aniontransportprotein Alpha-2-macroglobulin

164

6

15

Igmuchain C region

49

6.33

16 17

Actin, cytoplasmic 1 Ig alpha-1 chain C region

42 38

5.14 6.06

18

Filamin-A

283

5.64

19

Spectrinalphachain, erythrocytic 1 Ig gamma-3 chain C region

281

4.77

Band 3 aniontransportprotein Histidine-rich glycoprotein Actin, cytoplasmic 1 Ig gamma-2 chain C region Ig gamma-3 chain C region Haptoglobin

20

42

Igmuchain C region

7.79 Alpha-2-macroglobulin

516 188 53 79 102 61 42 37

IEP Fe3O4

MW [kDa]

IEP Fe3O4-NH2

MW [kDa]

IEP

61

7.1

37

5.86 Histidine-rich glycoprotein 8.18 Serum albumin

71

5.86

96

5.61 Plasminogen

93

6.91

8.25 Fibrinogen betachain

57

37

8.18

5.24 Fibrinogen gammachain 6.33 Histidine-rich glycoprotein 5.5 Apolipoprotein A-I 5.43 Serotransferrin 5.5 Ig gamma-2 chain C region 6.58 Apolipoprotein(a) 5.96 Complement C3 5.84 Haptoglobin

52

8.25 Ig gamma-1 chain C region 5.24 Complement factor H

143

6.18

61

7.1

Igmuchain C region

49

6.33

31 79 37

5.43 Fibrinogen alphachain 6.75 Fibrinogen betachain 7.44 Fibrinogen gammachain

96 57 52

5.61 8.25 5.24

5.5 Ig kappa chain C region 11 5.96 Apolipoprotein A-I 31 6.12 Ig gamma-3 chain C 42 region 6 Complement C3 188

5.5 5.43 7.79

5.5

515

5.5

516

6.58

79 37

6.75 7.44

38

6.06

515 188 45

6.75 Alpha-2164 macroglobulin 4.9 Ig kappa chain C 11 region 7.1 Apolipoprotein B-100 516

Apolipoprotein(a)

6.58 Apolipoprotein B-100

5.96

42

5.14 Igmuchain C region 49 7.44 Ig alpha-1 chain C 38 region 7.79 Complement factor H 143

6.33 Serotransferrin 6.06 Ig gamma-2 chain C region 6.18 Ig alpha-1 chain C region

45

6.12 Actin, cytoplasmic 1

42

5.14 Filamin-A

283

5.64

6

52

6.57 Band 3 aniontransportprotein

102

4.9

164

Hemopexin


MW [kDa]

83

84


Fig. 3. The Janus structure prevents the binding of coagulation proteins to NH2-functionalized iron oxide. Corona proteins identified by LC-MS and bioinformatically classified according to their biological activities in the blood system. Incubation time in human plasma as indicated (1 min vs. 60 min). For abbreviations see Supplementary Table 1.

workflow, we characterized the bound plasma components in detail, identifying and quantifying 176 proteins in the corona of the different nanoparticle formulations. We have previously shown that the protein corona evolves quickly and stays rather constant at prolonged exposure times [30]. Therefore, to investigate potential time-dependent changes in the protein corona, we performed mass spectrometric analysis of the protein corona at 1 min (as a model for early exposure times) and 1 h, as a model for prolonged exposure times. While we mostly did not observe major effects of the incubation time, significantly longer exposure times, or changes in the incubation temperature might modify the corona composition and therefore potentially affect particle stability in vivo. Strikingly, NH2 surface functionalized spherical particles (Fe3O4SiO2-PEG-NH2) displayed a strongly enhanced binding of the coagulation protein “histidine-rich glycoprotein” (HRG) and of complement factor H. This may lead to a stronger activation of the immune system in vivo. HRG is a plasma glycoprotein and regulates numerous biological functions and interacts with cell surface receptors and organizes immune clearance and coagulation [57]. High levels of HRG are associated with thrombotic disorders [70] suggesting pro-thrombotic effects of the Fe3O4-SiO2-PEG-NH2 nanoparticles. As the complement system participates in the elimination of pathogens [56], increased binding of complement factor H may increase clearance of the amine-functionalized Fe3O4SiO2-PEG-NH2. This steric “protection” by the hydrophobic Au

domain of the JPs blocks any polar interaction with the solution and prevents an increase of the hydrodynamic radius. The smaller amount of surface ligands (compared to the spherical particles) also prevents protonation of the terminal NH2 groups and a corresponding change of the zeta potential. Our data indicate that while spherical nanoparticles may be applied favorably for imaging, they may be less suited for drug loading due to a potential induction of an immune response. In contrast, NH2 groups at the Fe3O4 domain of the JPs did not lead to a significant difference in the distribution of the most abundant corona proteins, rendering Au-thiol@Fe3O4-SiO2-PEG-NH2 JPs more advantageous compared to spherical nanoparticles for drug delivery as well as imaging and therapy related applications. To correlate the corona data with the possible effects in vitro, we analyzed cellular ATP level as a marker of cell metabolism. Our results indicated an improved biocompatibility of both JPs (Authiol@Fe3O4-SiO2-PEG and Au-thiol@Fe3O4-SiO2-PEG-NH2) with a protein corona after incubation in human plasma. They were better tolerated than spherical nanoparticles with a corona and better than pristine JPs, indicating a better biocompatibility of coronacoated JPs. Furthermore, the generated corona leads to enhanced uptake for all nanoparticle formulations. The formation of reactive oxygen species caused by different nanoparticles resulted in apoptotic processes was established [71,72]. A high level of reactive oxygen species was also shown for spherical Fe3O4-SiO2-PEG in


85

Fig. 4. The protein corona enhances biocompatibility and uptake of Janus particles. (A) Relative cellular ATP levels were detected by ATPLite assay after 24 h. HMEC-1 treated with the different nanoparticles with or without a protein corona (PC). Data were normalized to control values (no particle exposure), which were set as 100% ATP level. *p < 0.05 (B) Uptake of nanoparticles into cells after incubation with 5 mg/mL Fe (II)/(III) for 24 h. Scale bars indicate 50 mm.

previous studies [15]. Interestingly, it was shown in literature that the gold domain of Au-thiol@Fe3O4-SiO2-PEG JPs exhibited an antioxidative potential against generated reactive oxygen species, thereby improving the biocompatibility of the JPs [15]. This may be especially important due to the higher cellular uptake of corona coated JPs compared to pristine JPs and control particles, which is in agreement with previous results showing an increased nanoparticle attachment and uptake into endothelial cells after corona formation [30]. The in vivo biodistribution of nanoparticles critically impacts

potential biomedical applications. However, only limited data regarding the in vivo application of gold at magnetite hybrid particles are available, especially regarding their cellular uptake and in vivo imaging abilities. Yang et al. previously developed Au@Fe3O4 hetero-nanostructures as affibody based trimodality nanoprobes (positron emission tomography, PET; optical imaging; and magnetic resonance imaging, MRI) for imaging of epidermal growth factor receptor (EGFR) positive tumors and have shown that they provided high specificity, sensitivity, and excellent tumor contrast for both PET and MRI imaging [73]. In our study, we comparatively

86


Fig. 5. Schematic representation of the in vivo experimental setup. The time points demonstrate injection of the respective probes, in vivo imaging and organ preparation. y: animals were sacrificed. N ¼ 9.

transported into the liver as determined by magnetic resonance imaging 24 h and iron content measurements 48 h post injection. Notably, despite PEGylation and the binding of serum albumin as a dysopsonin, which is known to prevent the detection of nanoparticles by phagocytic active cells [77], Au-thiol@Fe3O4-SiO2-PEG JPs were finally cleared by the body after 24 h. In summary, the formation of a plasma protein corona on JPs modified both size and surface charge. These changes affected both internalization into endothelial cells and cytotoxicity. Furthermore, the protein corona influenced the overall distribution throughout the body. Compared to spherical particles, JPs displayed an enhanced biocompatibility after uptake into cells. Importantly, the amphiphilic Janus structure could prevent the adhesion of opsonins to the NH2-groups indicating a prospective applicability for selected biomedical applications.

5. Conclusion investigated the biodistribution of Au-thiol@Fe3O4-SiO2-PEG JPs and spherical Fe3O4-SiO2-PEG control nanoparticles by MRI 24 h post injection and by atomic adsorption spectroscopy in mice 48 h post injection. Importantly, our data clearly demonstrated that the JPs described in this produce T2* -contrast in vivo. Previous studies investigating in vivo distribution and clearance of nanoparticles demonstrated the accumulation of 12 nm sized DMSA-coated magnetite nanoparticles after 3 h in the liver and spleen [74]. Other studies showed that 55% of radiolabeled USPIOS were found to accumulate in the liver already after 5 min and decreased to almost zero after 48 h, revealing an elongated blood life time supposedly caused by the PEGylation [75,76]. This is in agreement with our observations that 58% of the applied JPs were

To understand nano-biointeractions that occur after intravenous nanoparticle application, we investigated the behavior of Au-thiol@Fe3O4-SiO2-PEG Janus particles in a physiologically relevant environment. We demonstrated that protein coronas were generated rapidly. Protein corona composition was strongly affected by surface NH2-functionalization, while the Janus structure per se significantly reduced surface binding of coagulation proteins. JPs exhibit enhanced biocompatibility after internalization, whereas uptake was enhanced by the corona for all nanoparticle formulations. We demonstrated the general capability of JPs to generate T2* -contrast in the in vivo situation. Protein coronas depending on nanoparticle shape, size and charge have important implications for nanomedicine. Long-circulating nanoparticles are

Fig. 6. Au@Fe3O4 Janus particles generate similar T2*-contrast as compared to spherical Fe3O4 but offer the ability for multimodal imaging. (A) Magnetic resonance images of three representative mice for the different nanoparticle treatment groups 24 h post injection. Different tissues are marked as colored regions of interest: (i) The lung (yellow), muscle (blue), heart (red), (ii) liver (purple) and spleen (green). The white stripe in (i) indicate the sectional plane shown in (ii). In both, coronal and transversal slices the drastically reduced MRI signal in the liver and spleen is clearly visible (tissue is black) in comparison to the native images. At the same time other tissues (lung, muscle) show no changes in the MRI signal. (B) Atomic absorption spectrometry data of the isolated organs 48 h after nanoparticle application. N ¼ 9, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


necessarily macrophage-evading, requiring efficient strategies to design stealthy particles by preventing opsonization. The rich options for designing JPs provide ample opportunities to optimize particle characteristics for biomedical applications, e.g. by reducing side effects of amine-functionalization and enhancing potential anti-oxidative effects. Declaration of interest The authors declare no competing interests. Acknowledgments We acknowledge the German Research Foundation (DFG, HI689/8-2) for financial support of this project within the Priority Program PARCEL (SPP1313, HI-689/8-2).W.S. and S.T. were supported by Stiftung Rheinland-Pfalz (NANOSCH) and the DFG (SFB 1066); W.T. received funding by the DFG through SFB 1066. Isabel Schick is a recipient of a “Fonds der Chemischen Industrie VCI”Fellowship and is funded by the Excellence Initiative (DFG/GSC 266). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.07.049. References [1] T. Fujimoto, H. Ichikawa, T. Akisue, I. Fujita, K. Kishimoto, et al., Accumulation of MRI contrast agents in malignant fibrous histiocytoma for gadolinium neutron capture therapy, Appl. Radiat. Isot. 7e8 (2009) S355eS358. [2] D. Boyer, P. Tamarat, A. Maali, B. Lounis, M. Orrit, Photothermal imaging of nanometer-sized metal particles among scatterers, Science 5584 (2002) 1160e1163. [3] E.B. Dickerson, E.C. Dreaden, X.H. Huang, I.H. El-Sayed, H.H. Chu, et al., Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice, Cancer Lett. 1 (2008) 57e66. [4] X. Wu, T. Ming, X. Wang, P.N. Wang, J.F. Wang, et al., High-photoluminescence-yield gold nanocubes: for cell imaging and photothermal therapy, Acs Nano 1 (2010) 113e120. [5] N.J. Durr, T. Larson, D.K. Smith, B.A. Korgel, K. Sokolov, et al., Two-photon luminescence imaging of cancer cells using molecularly targeted gold nanorods, Nano Lett. 4 (2007) 941e945. [6] H. Jans, Q. Huo, Gold nanoparticle-enabled biological and chemical detection and analysis, Chem. Soc. Rev. 7 (2012) 2849e2866. [7] T.A. Taton, C.A. Mirkin, R.L. Letsinger, Scanometric DNA array detection with nanoparticle probes, Science 5485 (2000) 1757e1760. [8] J.F. Hainfeld, D.N. Slatkin, T.M. Focella, H.M. Smilowitz, Gold nanoparticles: a new X-ray contrast agent, Br. J. Radiol. 939 (2006) 248e253. [9] D. Xi, S. Dong, X.X. Meng, Q.H. Lu, L.J. Meng, et al., Gold nanoparticles as computerized tomography (CT) contrast agents, Rsc Adv. 33 (2012) 12515e12524. [10] Y.W. Jun, J.W. Seo, A. Cheon, Nanoscaling laws of magnetic nanoparticles and their applicabilities in biomedical sciences, Acc. Chem. Res. 2 (2008) 179e189. [11] S.S. Kang, G.X. Miao, S. Shi, Z. Jia, D.E. Nikles, et al., Enhanced magnetic properties of self-assembled FePt nanoparticles with MnO shell, J. Am. Chem. Soc. 4 (2006) 1042e1043. [12] S. Kossatz, R. Ludwig, H. Dahring, V. Ettelt, G. Rimkus, et al., High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature dosages in the tumor area, Pharm. Res. 31 (12) (2014 Dec) 3274e3288, http://dx.doi.org/10.1007/s11095-014-1417-0. Epub 2014 Jun 3. [13] T.D. Schladt, K. Schneider, H. Schild, W. Tremel, Synthesis and biofunctionalization of magnetic nanoparticles for medical diagnosis and treatment, Dalton Trans. 24 (2011) 6315e6343. [14] D. Yoo, J.H. Lee, T.H. Shin, J. Cheon, Theranostic magnetic nanoparticles, Acc. Chem. Res. 10 (2011) 863e874. [15] L. Landgraf, P. Ernst, I. Schick, O. Kohler, H. Oehring, et al., Anti-oxidative effects and harmlessness of asymmetric Au@Fe3O4 Janus particles on human blood cells, Biomaterials 25 (2014) 6986e6997. [16] I. Schick, S. Lorenz, D. Gehrig, A.M. Schilmann, H. Bauer, et al., Multifunctional two-photon active silica-coated Au@MnO Janus particles for selective dual functionalization and imaging, J. Am. Chem. Soc. 6 (2014) 2473e2483. [17] C. Xu, J. Xie, D. Ho, C. Wang, N. Kohler, et al., Au-Fe3O4 dumbbell nanoparticles as dual-functional probes, Angew. Chem. Int. Ed. 1 (2008) 173e176.

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