Stealth Iron Oxide Nanoparticles for Organotropic Drug Targeting

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Stealth iron oxide nanoparticles for organotropic drug targeting Massimiliano Magro, Davide Baratella, Emanuela Bonaiuto, Jessica De Almeida Roger, Giulia Chemello, Sonia Pasquaroli, Leonardo Mancini, Ike Olivotto, Giorgio Zoppellaro, Juri Ugolotti, Claudia Aparicio, Anna P. Fifi, Giorgio Cozza, Giovanni Miotto, Giuseppe Radaelli, Daniela Bertotto, Radek Zbo#il, and Fabio Vianello Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01750 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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Stealth iron oxide nanoparticles for organotropic drug targeting

Massimiliano Magro,† Davide Baratella,† Emanuela Bonaiuto,† Jessica de Almeida Roger,† Giulia Chemello,‡ Sonia Pasquaroli,‡ Leonardo Mancini,‡ Ike Olivotto,‡ Giorgio Zoppellaro,§ Juri Ugolotti § Claudia Aparicio,§ Anna P. Fifi,∥ Giorgio Cozza,# Giovanni Miotto,# Giuseppe Radaelli,† Daniela Bertotto,† Radek Zboril,*,§ and Fabio Vianello*,†

†Department

of Comparative Biomedicine and Food Science, University of Padua, Viale

dell’Università, Legnaro, 35020, Italy. ‡Department

of Life and Environmental Sciences, Marche Polytechnic University, via

Brecce Bianche, Ancona, 60131, Italy.


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§Department

of Physical Chemistry, Regional Centre of Advanced Technologies and

Materials, Palacky University in Olomouc, Šlechtitelů, Olomouc, 78371, Czech Republic. ∥BioTecnologie

BT S.r.l., Agrifood Technology Park of Umbria, Frazione Pantalla,

Pantalla, 06059, Italy. #Department

of Molecular Medicine, University of Padua, Viale G. Colombo, Padova,

35121, Italy.

ABSTRACT The ability of peculiar iron oxide nanoparticles (IONPs) to evade the immune system was investigated in vivo. The nanomaterial was provided directly into the farming water of zebrafish (Danio rerio) and the distribution of IONPs and the delivery of oxytetracycline (OTC) was studied evidencing the successful overcoming of the intestinal barrier and the specific and prolonged (28 days) organotropic delivery of OTC to the fish ovary. Noteworthy, no sign of adverse effects was observed. In fish blood, IONPs were able to


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specifically bind apolipoprotein A1 (Apo A1) and molecular modelling showed the structural analogy between the IONP@Apo A1 nano-conjugate and high-density lipoprotein (HDL). Thus, the preservation of the biological identity of the protein suggests a plausible explanation of the observed overcoming of the intestinal barrier, of the great biocompatibity of the nanomaterial and of the prolonged drug delivery (benefitting of the lipoprotein transport route). The present study promises novel and unexpected stealth materials in nanomedicine.

KEYWORDS: metal oxides, biomimetic nanomaterials, protein corona, biocompatibity, oxytetracycline delivery.


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INTRODUCTION Tuning the properties of nanostructured materials for applications in biological scenarios represents the current goal of the theranostics research. Envisioning the final extend of in vivo protein-nanoparticle interactions from the bench-synthesis is complicate,1 because an unavoidable array of possible unspecific interactions exists between the nanosystem and the biological molecules present in all living organisms.2, 3 The knowledge of the nature and strengths of these interactions and of their influence on the biological activity of the resulting nanobioconjugate constitute the essential know-how for the development of theranostic materials and drug carriers in biomedicine. Liposomes-encapsulated drugs and polymer–drug conjugates were the earliest nanotechnologies employed for biomedical applications, representing the foundation of advanced drug delivery.4, 5 Stimuli-responsive devices, including multiwalled carbon nanotubes and ultrasmall iron oxide nanoparticles, were proposed as new generation nanocarriers allowing the triggered release of the transported drug.6 Iron oxide nanoparticles have been studied in this context for decades, because they usually express an intrinsically low toxicity impact, making them compatible to be


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engineered for drug-delivery, as contrast agent materials and even tailored for medical therapies (hyperthermia). Years ago, our group developed a novel procedure for the synthesis of superparamagnetic iron oxide nanoparticles (IONPs), in the size range around 10 nm.7 The synthesis, completely carried out in water without the employment of any organic solvent or surfactant, leads to the formation of nanoparticles (maghemite phase, γ-Fe2O3), characterized by unique surface chemical behavior. These iron oxide nanoparticles, called SAMNs (surface active maghemite nanoparticles), display high water stability (e.g. colloidal suspensions) that does not require further surface modification or coating derivatization.8 These IONPs are a magnetic nanomaterial that shows great surface reactivity thanks to the presence of solvent exposed iron(III) atoms, and preserving the stable crystalline structure with high magnetic moment response (> 60 emu/g).7 From the combination of colloidal stability, excellent cell uptake,9 persistence within the host cells,10 low toxicity,9 and excellent MRI contrast agent properties,8 these nanoparticles can be considered elective carriers and flexible magnetic vectors for drug delivery. Moreover, as further distinctive tract, this nanomaterial, when exposed to biological fluids, possess the property of self-regulating


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protein absorption, leading to a protein corona composed of a selected and restricted group of bound proteins.9 This behavior, which is a rare example among nanostructured metal oxides, find an analogy with recent studies on poly ethylene glycol (PEG) coated nanoparticles, the current gold standard in drug delivery.11-14 The phenomenon was called “stealth effect”, and consists in avoiding the clearance of the drug vehicle by the immune system. It was believed that the stealth effect depends on the ability of PEG of preserving the nanocarrier surfaces from protein adsorption.15 Schöttler recently demonstrated that PEG coating on nanomaterials reduces the indiscriminate protein adsorption from the blood plasma thanks to the binding of specific proteins, such as apolipoprotein A1 (Apo A1) and clusterin.16 Accordingly, we already observed that when neat IONPs are introduced in calf serum, a small group of selected proteins, comprising apolipoproteins, were recognized and bound.9 However, the mere presence of the protein can not explain the stealth behavior of nanomaterials in biological systems.17 Herein, the selectivity and recognition properties of neat and oxytetracycline loaded IONPs for protein binding was firstly studied in fish blood, evidencing that apolipoprotein A1 was the prominent component of the resulting protein corona. Then, naked and OTC


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modified IONPs were followed in vivo in a fish animal model (zebrafish, Danio rerio). As in the case of PEG modified nanoparticles, IONPs seem to recruit specific proteins from the fish blood allowing their long term distribution within the host organism. Moreover, they likely benefitted of apoliproprotein A1 transport route for the targeting of the drug specifically to the ovary. In this view, computational modelling showed a structural analogy between the nanomaterial involving bound apolipoprotein A1 and high-density lipoprotein (HDL). Thus, the preservation of the biological identity of the protein and the consequent biomimetic features of the nano-bioconjugate provides consistent hints to explain the observed overcoming of the intestinal barrier, the great biocompatibity and the prolonged drug delivery of IONPs.

EXPERIMENTAL SECTION High-resolution Transmission electron microscope (HR-TEM) High-resolution Transmission electron microscope (HR-TEM) micrographs were acquired by a FEI Titan operating under an acceleration voltage of 80 kV with a point-topoint resolution of 0.08 nm. Before measurements, the aqueous suspension of the


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complex of magnetic nanoparticles and oxytretracycline (IONP@OTC, 0.4 mg mL-1) were diluted with double-distilled water (6 mL) and treated in ultrasound water-bath for 5 minutes. One drop (~10 µL) of the dilute suspension was placed onto a holey-carbon film supported by a copper-mesh TEM grid and dried in vacuum at room temperature. The transmission electron microscope (TEM) micrographs of biological samples were acquired by a FEI Tecnai 12 microscope operating at 120 kV.

Zeta-potentials and size-distribution Zeta-potentials and size-distribution of IONP@OTC and iron oxide nanoparticles were measured in water solutions at pH = 7.0 on a Zetasizer Nano particle analyser ZEN3600 (Malvern Instruments, UK) at T = 298 K. Triplicate experiments were performed. Statistical analysis on the size-distribution was obtained by using the LogNormalfunction.

Synthesis of iron oxide nanoparticles


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A typical synthesis of nanoparticles was already described7, 18 and can be summarized as follows: FeCl3·6 H2O (10.0 g, 37 mmol) was dissolved in MilliQ grade water (800 mL) under vigorous stirring at room temperature. NaBH4 solution (2 g, 53 mmol) in ammonia (3.5 %, 100 mL, 4.86 mol mol-1 Fe) was quickly added to the mixture. Soon after the reduction reaction occurrence, the temperature of the system was increased to 100 °C and kept constant for 2 hours. Then, the material was cooled at room temperature and aged in water, as prepared, for other 24 hours. This product was separated by imposition of an external magnet (NdFeB) and washed several times with water. This material can be transformed into a red brown powder (final synthesis product) by drying and curing at 400 °C for 2 hours. The resulting powder showed a magnetic response upon exposure to an external magnetic field. The final mass of product was 2.0 g (12.5 mmol) of Fe2O3 and a yield of 68 % was calculated. The resulting nanoparticulated material was characterized by Mössbauer spectroscopy, FT-IR spectroscopy, high resolution transmission electron microscopy (HR-TEM), XRD, magnetization measurements and resulted constituted of stoichiometric maghemite (γ-Fe2O3) with a mean diameter (davg) of 11 ± 2 nm, which can lead to the formation, upon ultrasound


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application in water (Bransonic, mod. 221, 48 kHz, 50 W) of a stable colloidal suspension, without any organic or inorganic coating or derivatization.7, 18 The surface of these bare maghemite nanoparticles shows peculiar binding properties and can be reversibly derivatized with selected organic molecules. We called these iron oxide nanoparticles as Surface Active Maghemite Nanoparticles (SAMNs). SAMNs are currently produced and delivered by AINT s.r.l. (Venice, Italy).

Oxytetracycline binding on iron oxide nanoparticles Oxytetracycline (20 mg L-1) was incubated in the presence of various nanoparticle concentrations (0.1, 0.2, 0.5, 1.0, 1.5 and 2.0 g L-1) in H2O at room temperature. Nanoparticle suspensions were previously treated by ultrasound (Bransonic, mod. 221, 48 kHz, 50 W) for 5 minutes, and then stored, under overnight constant agitation, to promote complex formation. Then, magnetic nanoparticles were separated from the aqueous solution with the assistance of a magnet (N35, 263-287 kJ m-3 BH, 1,1701,210 mT flux density by Powermagnet, Germany) for 1 hour. IONP@OTC was washed


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several times under constant agitation. Oxytetracycline concentrations in the supernatants were probed by UV-Vis spectrophotometry.

Animals and housing Four hundred fifty female zebrafish (Danio rerio), AB strain, were maintained in four 100 L tanks equipped with biological, mechanical and UV filtration (Panaque, Italy) at 28°C and subjected to a 12L:12D photoperiod. Ammonia and nitrite were constantly kept below 0.01 mg L−1. Water temperature, dissolved oxygen, pH, ammonia and nitrite levels were monitored daily. All procedures involving animals were conducted in line with Italian legislation on experimental animals and were approved by the Health Ministry’s department of Veterinary Public Health and by the ethics committee of Università Politecnica delle Marche (Authorization N 640/2015-PR). Optimal rearing conditions were applied throughout the study, and all efforts were made to minimize animal suffering by using an anesthetic (MS222; Sigma Aldrich).

Fish exposure to OTC and IONP@OTC


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At the beginning of the experiment, fishes were randomly distributed in 9 tanks (100 L) and treated as follows for 28 days: Group A: control. Three 100 L tanks with 50 fish each; Group B: Three 100 L tanks added with OTC dissolved into the water (4 mg L-1) with 50 fish each; Group C: Three 100 L tanks added with 100 mg L-1 of IONP@OTC (corresponding to 4 mg L-1 of OTC) with 50 fish each. Fish sampling was performed at day 0, 14 and 28 after the experiment started. Livers, ovaries and intestines were collected and stored at -80°C for further analysis. All fish were anesthetized with a lethal MS222 (Sigma Aldrich) dose. Each week 3 × 50 mL water samples were collected from the 9 tanks in order to evaluate OTC concentrations during the experiment.

Incubation of iron oxide nanoparticles and IONP@OTC in fish plasma Iron oxide nanoparticles or IONP@OTC (50 µg) were incubated in fish (gilthead bream,

Sparus aurata) blood serum (200 µL, 20 mg mL-1 average protein concentration) for 2 hours under end-over-end mixing. After incubation, magnetic nanoparticles were separated by the application of an external magnet, the supernatants were removed,


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and, finally, magnetic nanoparticles bearing bound proteins were subjected to 2 washing cycles (200 µL) with water. Bound proteins were released from nanoparticle surface by treatment at 95°C (5 min) in 250 mM TRIS, pH 8.5, 2% SDS, 50 mM DTT. Released proteins were analyzed by SDS gel electrophoresis. Most intense electrophoretic band was excised and digested by trypsin, and proteins were identified by mass spectrometry.

Homology modelling Gilthead bream and zebrafish Apo A1 models were built using a homology modeling approach (under Amber99 force field) implemented into Molecular Operating Environment (MOE, Chemical Computing Group3) with three different crystal structures of the homologous human Apo A1 as template (PDB code: 2AO1, 1AV1 and 3R2P). The alignments between the templates and the target sequences were performed using the Protein Align tool implemented in MOE, in particular the Needleman-Wunsch methodology with Blosum 50 matrix was used. All the ligands and cofactors were removed from the template structures; for each model, 10 intermediates were built, and


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the final models were based on the best-scoring intermediate model ranked by the electrostatic solvation energy, calculated used a Generalized Born/Volume Integral (GB/VI) methodology in MOE. The final models were subjected to the Protonate 3D application to optimize ionization states and proton placement (MOE). Finally, a refinement step (called “medium refinement”) has be applied to the final models to relieve steric strain and moderately relax the final structures.

Protein-protein docking To study the multi structure configurations (dimer, trimer and tetramer) of the gilthead bream and zebrafish Apo A1, Protein-Protein docking analysis was performed using two FFT-based docking software PIPER. Intermediate complexes (n = 1,000) were obtained from PIPER algorithm; the final complex was chosen according to electrostatic solvation energy scoring function and to human homologous Apo A1 crystal structures representing multimeric geometry, by selecting complexes with an RMSD (Root Mean Square Deviation) < 3Å compared to the crystallographic ones.19


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SDS-PAGE electrophoresis 1-D SDS-PAGE were performed using Bis-Tris 4-12% polyacrylamide gels (NuPAGE, Invitrogen) with MES buffer, at 160 V constant tension for approximately 1 h (until the front line reached the end of the gel). Samples were dissolved in a solubilizing buffer (10% glycerol, 250 mM TRIS, pH 8.5, 2% SDS, 50 mM DTT, Coomassie Blue), heated for 10 min at 70°C, centrifuged for 1 min and then loaded onto the gel, together with molecular mass weight standards. After SDS-PAGE runs, gels were stained with Colloidal Coomassie and finally gel images were acquired by a digital equipment (Kodak 4000MM).20

In gel digestion of proteins Selected protein bands were manually excised from the gel and protein digestion was performed in gel, using sequencing grade modified trypsin (Promega, Madison, WI). Briefly, excised gel bands were repeatedly washed with 50% acetonitrile/40 mM NH4HCO3 and then dried under vacuum. Trypsin (20 µL, 12.5 ng µL-1 in 40 mM NH4HCO3) was added to each gel band and samples were incubated for 30 min at 4°C.


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Then, excess trypsin was discarded and replaced by 25 µL of 40 mM NH4HCO3, containing 10% acetonitrile. Digestion was carried out at 37°C, overnight. After trypsin digestion, peptides were extracted from the gel with 50% acetonitrile/0.1% formic acid (25 µL), repeated twice. The final peptide mixtures were then dried under vacuum and finally re-suspended with of 5% acetonitrile/0.1% formic acid (20 µL).

Protein identification by mass Spectrometry (MS) All samples were analyzed by LC-MS/MS using a 6520 Q-TOF mass spectrometer, coupled on-line with a 1200 series HPLC system through a Chip Cube Interface (Agilent Technologies, CA, USA). Each sample (4 µL) was loaded onto a C18 large capacity chip-column, integrating a 300 nL capacity trap-column, a RP column (75 µm × 150 mm), connection capillaries, and a nano-spray emitter. Solvent A was water containing 0.1% formic acid, while solvent B was acetonitrile containing 0.1 % formic acid. Peptides were separated with a linear gradient of 0 – 50% of solvent B in 50 min at a flow rate of 0.3 µL min-1. Mass spectra were acquired in a data dependent mode: MS/MS spectra of the 4 most intense ions were acquired for each MS scan in the 140 –


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1,700 Da range. Scan speed was set to 3 MS spectra s-1 and 3 MS/MS spectra s-1. Capillary voltage was set to 1,720 V and drying gas to 4 L s-1. Raw data files were converted into Mascot Generic Format (MGF) files with MassHunter Qualitative Analysis Software (Agilent Technologies). MGF files were analyzed using Mascot Search Engine, server version 2.3 (Matrix Science, London, UK). Spectra were searched against the SwissProt database (May 2011 version, Taxonomy Mammalia, 65,453 entries) with the following parameters: enzyme specificity was set to trypsin with up to 2 missed cleavages, peptide and fragment tolerance were set to 6 ppm and 0.05 Da respectively. Deamidation (NQ), phosphorilation (ST), oxidation (MC) and propionamide (C) were selected as variable modification. Proteins were considered as positively identified if at least 3 peptides with individual significant score (p < 0.05) were sequenced. The search was carried out also against the corresponding randomized database, which did not return any positive identification under the same strict conditions (i.e., at least 3 peptides sequenced with individual significant score).

Determination of OTC in biological samples by HPLC-MS


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About 100 mg of each tissue sample were weighed, triturated and homogenized in a blender, placed in a plastic tube and dissolved in 1 ml of ultrapure water acidified with 1% phosphoric acid. The tube was vigorously mixed for 2 minutes, treated in ultrasonic bath for 5 minutes and then centrifuged at 13,000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.2 µm membrane filters and stirred before injection into the chromatograph. A 20 µL aliquot was injected. The separation conditions for the OTC were a mobile phase flow of 0.8 mL min-1 containing acetonitrile acidified with 0.1% phosphoric acid and ultrapure water acidified with 1% phosphoric acid (15:85, v/v) filtered through a 0.45 µm nylon filter under vacuum and degassed by ultra-sonication. Column oven temperature was 25°C and ultraviolet detector operated at wavelength of 360 nm. The quantitation was accomplished by using an analytical calibration curve built with six concentration levels in the range 20 – 250 µg L-1 (OTC). Extraction recoveries were determined by spiking untreated biological samples (100 mg) with a fortification solution at LOQ and LOD levels: 500 and 100 µg kg-1.


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The HPLC system (Agilent 1100 series with Chemstation) used for analysis consists of a quaternary pump (Agilent 1100 series), a vacuum degasser, an injector and a wavelength ultraviolet detector (Agilent 1100 series). The chromatographic column was an analytical reversed-phase Zorbax Eclipse XDB C18, 4.6 × 150 mm, 5 µm. The method was “in-house” validated using the following performance criteria: linearity, sensitivity and selectivity, detection and quantification limits, accuracy and precision. The solution for calibration and fortification were prepared in ultrapure water acidified with 1% phosphoric acid and stored at temperature ≤ -18 °C. Linearity, sensitivity and selectivity, detection and quantification limits were established by the analytical curve (20, 40, 50, 80, 100 and 250 µg L-1 of OTC). The limit of quantification (LOQ) was obtained from the calibration curve. The limit of quantification (LOQ) was expressed in µg/kg by the following formula: OTC (µg L-1) × Vex (mL) / W (g), where Vex is the extraction volume and W is the untreated sample weight. The limit of detection (LOD) was the lowest amount of an analyte in a sample that can be detected but not necessarily quantitated as an exact value and it was 100 µg kg-1. The precision of the method, expressed as the relative standard deviation of peak area measurements


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(n = 5), was evaluated by the recovery data obtained (accuracy) at the LOQ concentration level (RSD% = 6.51). The accuracy of the method, expressed as percent recovery, was determined at 500 µg kg-1 that was the LOQ concentration level (Mean Recovery 95.5%).

RESULTS Selective protein binding to IONPs in fish blood serum Upon introduction in a biological fluid, nanomaterials are rapidly coated by a number of proteins, leading to a protein corona that governs the behavior and the fate of the nanomaterial in the biological system. With the final aim to test IONPs in vivo in zebrafish (Danio rerio) as animal model and considering that most fish plasma proteins are well conserved, the protein corona composition on neat iron oxide nanoparticles was evaluated upon incubation in blood serum from a larger fish (Sparus aurata, gilthead bream) (n = 5) and compared to oxytetracycline (OTC) modified IONPs (IONP@OTC). IONP@OTC was self-assembled as follows: (i) mixing together a water suspension of neat iron oxide nanoparticles and OTC for 5 minutes, (ii) storage


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overnight under constant agitation, and (iii) magnetic separation of the final core-shell nano-composite. The hybrid core-shell nanomaterial was characterized by measuring the zeta-potential (), at room temperature and at pH = 7.0, and by transmission electron microscopy. Following the binding of oxytetracycline by the iron oxide nanoparticles, a substantial reduction of  is witnessed, from +38.7 ± 8.7 mV for neat iron oxide nanoparticles (conductivity 0.00347 mS cm-1) down to +6.7 ± 2.3 mV for IONP@OTC (conductivity 0.0146 mS cm-1). An extensive characterization of IONP@OTC is presented in Supporting Information and in Figure 1.


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Figure 1. Characterization of IONP@OTC complex. Panels A and D show the HR-TEM image of the neat IONPs and panel B the STEM-HAADF image of neat IONP. The Electron dispersive spectroscopy (EDS) mapping of IONP is shown in panels C, E and F with the distribution of oxygen (panel C), iron (panel E) and the elemental overlay (panel F). Panels G and L show the HR-TEM images of IONP@OTC nanoparticles with the STEM-HAADF image (panel H). Electron dispersive spectroscopy (EDS) mapping of IONP@OTC showing the distribution of oxygen (panel I), iron (panel J), nitrogen (panel K), carbon (panel M) and the elemental overlay of Fe,N,C (panel N). Size bars in H, I, J, K, M and N correspond to 10 nm. Panel O shows the optimized DFT structure of the oxytetracycline molecule (RBP86/6-31G*, neutral form, gas phase) with the labelled A, B, C, D ring-chromophores. Panel P shows the comparison of the FT-IR spectrum of neat IONP (upper yellow-trace) and IONP@OTC (lower red-trace). Panel Q shows the powder XRD pattern (Co-Kα radiation) of IONP@OTC nanoparticles. The numbers are the Miller indices for the maghemite phase. The hematite contribution (marked by *) is found below 5 wt %.


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The morphology of the nanomaterial upon protein corona formation can be appreciated in HR-TEM micrograph (see Figure S12D). The nature and the amount of absorbed proteins on nanoparticle surface was determined by gel electrophoresis and mass spectrometry, as described in the Experimental Section. Interestingly, the electrophoretic gel of proteins from the surface of iron oxide nanoparticles and IONP@OTC showed only one intense band at about 60 kDa and few minor components. This electrophoretic band was excised and digested by trypsin, and proteins were identified by mass spectrometry (LC/MS/MS as described in the experimental Section). Mass spectrometry substantiated the presence of apolipoprotein A1 (Apo A1, 28 kDa) as dominant component of protein corona.19 Along with clusterin, Apo A1 represents the main protein bound to stealth polymers16 and supposed to camouflage nanoparticles from the immune system clearance. In addition, Apo A1 is a known carrier for lipids and low polarity molecules to ovary in fishes. In fact, during vitellogenesis, Apo A1 is sequestered via receptor-mediated uptake by the developing oocytes in the fish ovary.21 Notably, Apo A1 is synthesized and assembled in the liver and in the intestine, before being secreted in the plasma.


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As the orientation of bound proteins on nanoparticle surface is of fundamental importance for the following fate of the nano-conjugate in the biological system,22 Apo A1 binding on IONPs was analyzed by homology modeling, as described in the Experimental Section. The structural models of Apo A1 of gilthead bream (Sparus

aurata), zebrafish (Danio rerio), and human (Homo sapiens) were built for comparison. Carboxylic groups, belonging to aspartic acid and glutamic acid residues, were considered for their ligand properties toward iron chelation and for their abundance in proteins.23 Similarly to phosphate groups in nucleic acids,24 the affinity of proteins for naked IONPs has been explained in term of distribution of carboxylic groups matching the surface topography of under-coordinated iron(III).25 Thus, the regions of the

Apo

A1 surface presenting the highest density of carboxyl distribution were evidenced. The dimeric structures of Apo A1 from gilthead bream and zebrafish, built in accordance with the geometry of the crystal structure of human homolog (3R2P),26 are characterized by a semicircular backbone formed from antiparallel helical repeats. It must be noted that an extended negatively charged distribution is located on the internal face of the halfcircle of both gilthead bream and zebrafish Apo A1 (see Figure 2A, structure a and b).


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Figure 2. Representation of the binding of a dimer of gilthead bream Apo A1 on an iron oxide nanoparticle and gel electrophoresis of proteins bound on iron oxide nanoparticles after incubation in plasma from gilthead bream (Sparus aurata). (A) Apo 1 dimers (a, c) are shown as analytic Connolly’s electrostatic surface charge distribution, calculated using AMBER99 Forcefield. Blue color indicates positive surface charges and red color indicates negative surface charges. The Apo A1 monomer is composed of four antiparallel helixes at N-terminal and two antiparallel helixes at C-terminal domains. Apo 1 dimer bound on iron oxide nanoparticle (b). (B) Electrophoretic profile of proteins released from the surface of iron oxide nanoparticles and IONP@OTC. Proteins bound to iron oxide nanoparticles or IONP@OTC were released by 2 M NH4OH. The most


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intense bands (at about 60 kDa, marked with an arrow in the gel photograph) were analyzed by LC/MS/MS after tryptic digestion and resulted Apo A1.

Intriguingly, the diameter of the half-circle is 9.5 nm is compatible with the diameter of iron oxide nanoparticles, plausibly approximated to a sphere. Thus, we suggest that the dimer of Apo A1 from gilthead bream and zebrafish binds to nanoparticles in a sort of belt mode (see Figure 2A, structure c), taking into account the flexibility of Apo A1 structure reported elsewhere.26 In this model, the negatively charged aminoacids located in the internal face of the belt structure directly interact with solvent exposed iron(III) sites on nanoparticles surface, leading to an extended multiple point binding. This concept is in agreement with the crystal structure of human Apo A1 (3R2P) representing a half-circle flexible dimer implicated in the formation of discoidal highdensity lipoprotein (HDL) particles.26, 27 It should be mentioned that HDL particles are often composed of two to four molecules of Apo A1.28 Hence, we do not exclude the possibility that other superstructures of gilthead bream or zebrafish Apo A1, such as trimers and tetramers, could possibly interact with IONPs in vivo.


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In order to explore the Apo A1 superstructures presenting suitable features for the binding to iron oxide nanoparticles, homology modeling technique was employed (see Experimental Section) and the architecture proposed for the human Apo A1 crystal structure was followed by a protein-protein docking approach. The crystal structure templates (2A01, 1AV1 and 3R2P) were retrieved from PDB (Protein Data Bank), representing human Apo A1, in three different states, lipid free, lipid bound and HDL assembly conformations, respectively.28-30 The structure in the apo-form (2A01) is described as a trimer composed of three Apo A1 monomers bound in a threefold structure around a D3 symmetric Cr(III)-tris-acetylacetonate (Cr-acac3) moiety.27 Contacts between Apo A1 and Cr-acac3 are critical to maintain the trimer structures.27 Human Apo A1 monomer displays a large negative pattern with a high concentration of carboxyl side chains exposed to water in a α-helix secondary structure.27 A similar negatively charged distributions is present on the gilthead bream and zebrafish Apo A1 monomers (Figure 3A), and available to interact with iron oxide nanoparticles.


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Figure 3. Analytic Connolly’s electrostatic surface charge distribution of Apo A1 calculated using AMBER99 Forcefield. Blue color indicates positive surface charges and red color indicates negative surface charges. (A) Apo A1 monomers: human (1), gilthead bream (2) and zebrafish (3); (B) Apo A1 trimeric structures: gilthead bream (1) and zebrafish (2); (3) graphical representation of the interaction between Apo A1 trimer and iron oxide nanoparticle (brown). (C) Graphical representation of the “belt mode” tetrameric structure of Apo A1: gilthead bream (1) and zebrafish (2).

Actually, the trimeric structure of Apo A1 from gilthead bream and zebrafish, derived from the crystallographic trimeric assembly of the human homolog,27 shows unique negative charge distributions (Figure 3B). In fact, the carboxylic acid distribution in the threefold structure of gilthead bream and zebrafish Apo A1 is uniformly distributed in a


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superstructure, starting from the center and extending along three braces (Figure 3B, structure 1 and structure 2). This suggests that both the threefold structures display a regular and continuous negative surface able to interact with spherical objects of about 10 nm. This is particularly true considering one of the two faces of the trimeric structure, presenting the highest concentration of negatively charged aminoacids (Figure 3B structure 2). Interestingly, this face presents a concave surface, 4 nm deep, perfectly compatible for interacting with a single iron oxide nanoparticle with a diameter of 11 nm. Even if it has been noted that the trimeric structure of Apo A1 could be enhanced in the crystal structure by the presence of Cr-acac3 molecules,27 we cannot exclude that iron oxide nanoparticles could force the formation of Apo A1 trimeric structure, as described above, leading to the interaction mode suggested in Figure 3B structure 3. The human Apo A1 lipid bound structure (1AV1) is formed by four Apo A1 molecules constituted of a continuous amphipathic α-helix and associated via hydrophobic interactions to form an antiparallel four-helix bundle with an elliptical ring shape;31 this structure is ideal for wrapping around either discoidal or spherical HDL.29 To note that HDL particles are constituted of two or four Apo A1 molecules;28 these findings are in


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agreement with the third crystal structure of human Apo A1 (3R2P) considered in the present study. Thus, the geometry of the Apo A1 tetramer models from gilthead bream and zebrafish presents an extended negatively charged surface distributed in an antiparallel four-helix bundle with an elliptical ring shape (Figure 3C), as a natural evolution of its dimeric structure. As in the case of the dimeric structure of Apo A1, also the tetrameric ellipse is available for the direct interaction with iron oxide nanoparticles. In fact, the flexible elliptical ring shape measures 12.5 × 8.0 × 4.0 nm, compatible with a complete winding over the iron oxide nanoparticle, as proposed for HDL particles formation.27 Hence, the presence of Apo A1 as dominant component of protein corona on IONPs may plausibly provide biomimetic properties. On these basis, as Apo A1 could act as an endogenous targeting functionality, the analogy with HDL could be the key for interpreting the transport of the Apo A1 coated IONPs from the intestine to the ovary, as hereafter observed. Thus, neat and OTC modified nanoparticles were tested in living zebrafish (Danio rerio) by addition to the farming water.

IONPs adsorption and OTC delivery in zebrafish (Danio rerio)


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As protein corona formation determines the fate of nanoparticles into an organism,32,33 defining their stealth properties and targeting ability, IONPs behavior was investigated in

vivo. Neat IONPs and IONP@OTC (as drug carrier) were tested on an aquatic animal model (Danio rerio, zebrafish). An experiment involving 150 animals for each treatment (in triplicate) was carried out, providing 4 mg L-1 OTC and 100 mg L-1 IONP@OTC (corresponding to 4 mg L-1 OTC) in the farming water. The concentration of OTC was followed in animal intestine, liver and ovary by HPLC-MS at 14 and 28 day treatment in order to evaluate drug bioavailability. Noteworthy, two different distribution scenarios emerged from free or IONP immobilized OTC administrations: the binding of OTC on nanoparticle surface enabled the overcoming of the intestinal barrier and its bio-distribution within the host organism, leading to its accumulation in the ovary. Conversely, the antibiotic was confined in the intestinal tract in animals treated with free OTC, being the organ in direct contact with the environment, and negligible concentrations were found in the ovary. Upon IONP@OTC administration, the concentration of OTC in the intestine and ovary were


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superimposable (10 mg kg-1) and resulted constant over 28 days. Besides witnessing OTC absorption by the intestine, the specific organotropic drug delivery envisages that nanoparticles benefitted of the lipoprotein transport route. In order to provide a full insight into the fate of IONPs into the host organism, nanoparticle distribution was studied by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), in combination with optical and electron transmission microscopies (TEM). The whole body iron concentration in the population treated with iron oxide nanoparticles (97.7 ± 5.8 and 96.6 ± 9.8 µg g-1, in IONP and IONP@OTC treated animals, respectively) resulted higher than the basal content of the metal in controls (16.6 ± 2.6 µg g-1). Moreover, ICP-AES indicated that 50 % of the whole iron content in animals treated with IONPs and IONP@OTC overcame the intestinal barrier, and the rest was in the intestine. In this organ, iron concentration was twenty times higher (574.4 ± 38.1 and 503.6 ± 74.9 µg g-1, in IONP and IONP@OTC treated animals, respectively) than in controls (27.4 ± 7.9 µg g-1). Noteworthy, iron content in skin and in


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gills, potential paths for nanoparticles adsorption, were comparable in treated and control animals. Electron microscopy images (Figure 4, panels A-D) of the intestinal region of treated animals evidenced that IONPs were present solely as isolated nanoparticles and were adsorbed on the surface of villi and inside enterocytes.

Figure 4. Optical micrographs and electron microscopy images of zebrafish intestinal tissue treated with IONP@OTC. (A) Photomicrograph of cross-section of intestine


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showing intestinal villi in zebrafish treated with 100 mg L-1 IONP@OTC. Each villous is lined by simple columnar epithelium containing absorptive cells with uniform and continuous striated border of microvilli and lamina propria in the core of each villous. No macroscopic aggregates of nanoparticles are detectable. H&E staining. (B, C) Transmission electron microscopic detection of single electron-dense IONP@OTC attached to the surface of microvilli (B, C: arrows). Structures resembling nanoparticles were also observed inside gut cells (B: arrows). (D) Higher magnification of the same region (C) revealing single spherical nanoparticles (arrows) attached to the apical region of microvilli.

Nanoparticle adsorption appeared homogenous in the whole tissue and no sign of morphological damage to enterocytes was observed, conversely to other reported nanomaterials, such as gold,35 TiO2 36-38 and silver.39 Differently from other nanosystems40 and in contrast with the massive nanoparticle adsorption by the intestinal tract witnessed by ICP-AES and HR-TEM, no sign of inflammatory response, such as lymphocytes and granulocytes infiltration, was observed with IONPs and


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IONP@OTC. Furthermore, optical micrographs confirmed the absence of macroscopic nanoparticle aggregates in the intestine (Figure 4A). In fact, Philbrook and collegues correlated the formation of large aggregates of carbon nanotubes with the loss of crossing efficiency through intestinal cells.41 Therefore, nanoparticles in the farming water entered in the fish gastro-intestinal tract and were easily adsorbed with no sign of adverse effects, prompting a stealth effect occurrence. The liver and the ovary of zebrafish treated with IONPs and IONP@OTC were explored by optical and electron microscopy and representative images are reported in Figure 5.


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Figure 5. Optical micrographs and electron microscopy images of zebrafish liver and ovary tissues treated with IONP@OTC. (A) Photomicrograph of cross-section of liver parenchyma in 100 mg L-1 IONP@OTC administered zebrafish, showing the organization in hepatocytes cells. No clusters of nanoparticles are detectable. H&E staining. (B) Transmission electron microscopic detection of hepatocyte cells surrounding a capillary. (C) Higher magnification of the same region (B) reveals single spherical nanoparticles (arrows) inside the capillary. (D) Transmission electron


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microscopic detection of nanoparticles in the space between the follicular epithelium and the zona radiata of zebrafish ovary.

Nanoparticle aggregates were absent in the liver parenchyma (data confirmed by ICPAES), which showed no sign of alteration with respect to controls (Figure 5A). Interestingly, free-circulating IONP@OTC and IONPs (or small nanoaggregates) can be observed in small liver blood vessels (Figure 5 B and C). Moreover, unmodified iron oxide nanoparticles, as well as, the drug carrier were evidenced in the ovary (Figure 5D). It should be noted that nanoparticle concentration, expressed in term of iron content, was constant in explored organs over a period of 28 days. Finally, control experiments treating zebrafish with 100 mg L-1 iron oxide nanoparticles, showed no indications of acute or long term (28 day) toxicity, ruling out possible concerns about in vivo applications of iron oxide nanoparticles on this animal model. The combination of nanomaterial biocompatibility and easy absorption by the animal intestine suggests that the administration of IONPs through farming water leads to the overcoming of biological barriers and to an improvement of the known low bioavailability


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of the OTC,42 aspects of particular relevance for a therapeutic strategy.43-45 In fact, IONP@OTC can be of interest for treating specific vertically transmissible bacterial infections in fishes, such as those involving Bacterium salmoninarum and

Flavobacterium psychrophilum affecting aquacultured trouts.46-50

DISCUSSION It is widely accepted that stealth effect represents the resistance against uncontrolled protein absorption on nanomaterials exerted by coating compounds. On these basis, by controlling the surface chemistry of nanoparticles, one can tailor the protein corona for the best affinity and specificity for peptides and proteins aimed at new targeting opportunities. We developed peculiar iron oxide nanoparticles (IONPs) which are able to specifically bind proteins presenting high affinities for their surface during the initial period of incubation in a biological fluid, even if present in very tiny concentrations. This distinctive nanoparticle surface, being constituted of common maghemite (γ-Fe2O3), is the result of the unusual crystal organization at the boundary with the solvent, that, in


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some extent, creates a selective and compatible interface for protein docking.51 Noteworthy, among the proteins present in fish blood, IONPs, either as such or modified with an antibiotic (oxytetracycline, OTC), were able to specifically bind apolipoprotein A1. Apolipoprotein A1 is normally sequestered via receptor-mediated uptake by the developing oocytes in the fish ovary, and it can be therefore assumed as an endogenous targeting signal. Thus IONPs, neat or loaded with OTC, were tested in vivo in an animal model (Danio rerio, zebrafish). The fast intestinal absorption, the absence of any sign of adverse effect on animal tissues and the extended organotropic drug delivery offered consistent hints on the stealth behavior of the nanomaterial. Indeed, nanoparticles, likely benefitting of apolipoprotein A1 transport route, avoided unspecific protein absorption and immune system clearance, providing a targeted delivery of OTC to the ovary. The role of apolipoprotein A1 in determining the stealth properties of polymeric coatings of nanoparticles, such as poly-ethyl ethylene glycol (PEG) or poly-ethyl ethylene phosphate (PEEP) was demonstrated.16 However, the mere presence of the protein does not explain the stealth behavior of the nanomaterial in a biological system. In fact,


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several nanoparticles were reported to display significant amounts of Apo A1 in their protein corona upon exposure to physiological environments.17 Other factors should be taken into account, such as the preservation of protein tertiary structure and orientation upon binding. Indeed, the comprehension of functional biomolecular motifs at the biointerface of the protein corona, which allow the recognition by cell receptors or biological barriers, is of crucial importance in the view of nanomaterial engagement with biological pathways.52,53 In the present work, the dimer of Apo A1 was found as the predominant component of the protein corona on IONPs, demonstrating very high affinity for the nanomaterial surface. Computational modelling explained the structural reasons behind the selective binding of Apo A1 to IONPs and described the role of the proper distribution of carboxylic groups and elongated shape of the protein, which lead to the ready interaction with IONPs surface. Noteworthy, as already observed,9 proteins readily interacting with IONPs do not undergo drastic modifications of their structure for adapting to nanoparticle surface. Molecular modelling showed the structural analogy between the IONP@Apo A1 nano-conjugate and high-density lipoprotein (HDL). Thus,


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the concept of preservation of the biological identity of the protein helps in explaining the observed overcoming of the intestinal barrier, the great biocompatibity of the nanomaterial and the prolonged drug delivery. To the best of our knowledge, no other examples of stealth effect governed by the spontaneous binding of specific proteins on a metal oxide surface have been so far reported. The present study offers an alternative viewpoint on protein corona formation on nanomaterials, from a detrimental uncontrollable phenomenon to new targeting opportunities. Moreover, this work suggests to open new doors in the classical view on the stealth effect in nanomedicine, offering a contribution to the nascent knowledge on the ability of nanoparticle chemistry to control the fate of drug vehicles inside the organism.

CONCLUSIONS Stealth effect, namely the ability of nanomaterial coatings to preserve nanovehicles from immune system clearance, was lengthily believed to be a prerogative of polyethylene


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glycol or similar polymer materials. It was interpreted as the property of the coating to be an antagonist toward protein corona formation. Recently, this idea was revolutionized by the demonstration that stealth effect is the consequence of the formation of a shell of selectively bound apolipoproteins. In the present manuscript, the general validity of this concept was corroborated by testing in vivo a pristine nanostructured iron oxide with exquisite selectivity toward proteins. The nanomaterial was able to evade the clearance of zebrafish immune system, as witnessed the massive absorption from the intestinal tract, with no sign of adverse outcome, and by the organotropic delivery of OTC antibiotic. The present work helps to open a new avenue to the evaluation of novel stealth nanomaterials for biomedicine.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Supporting Results (Self-assembly of oxytetracycline on iron oxide nanoparticles, chemical characterization of IONP@OTC) Supporting Figures (structure and optical


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spectrum of OTC, decay of OTC in water, amount of bound OTC on nanoparticle surface, TGA and EGA analysis, FT-IR, electrostatic potential surface of OTC, XPS, UV-Vis spectroscopy, Mössbauer analysis, HR-TEM micrographs of IONP@OTC), Supporting Tables (Standard thermodynamic quantities of OTC, growth of E. coli in LB medium with OTC), Supporting References and Supporting Equation (saturation function of nanoparticle surface fractional coverage) (PDF).

AUTHOR INFORMATION

Corresponding Authors *Prof. Fabio Vianello, E-mail: [email protected], Phone: (+39) 0498272638, Fax: (+39) 0498272973. Prof. Radek Zboril, E-mail [email protected], Phone: (+420) 58 563 4337, Fax: (+420) 58 563 4958.

Funding


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This work was supported by CAPES (Brazil), process n. 13160-13-3, University of Padua (Italy), grant “Assegni di Ricerca Junior” 2014 n. CPDR148959, CARIPARO Foundation and the excellence department project of the Italian Ministry of Education, University and research (MIUR) "Centro di Eccellenza per la Salute degli Animali Acquatici - ECCE AQUA". Moreover, authors gratefully acknowledge the support of the Nanomaterials and Nanotechnologies for Environment Protection and Sustainable Future (NanoEnviCz, LM2015073) and the project LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic. The authors thank the electron microscopy facility of the Biology Department and the Molecular Modelling Section of Padua University. Finally, authors would like to thank Biotecnologie BT S.r.l. for supporting a GC PhD.

Notes The authors declare no conflict of interest.


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ACKNOWLEDGMENT Authors gratefully acknowledge CAPES (Brazil), process n. 13160-13-3, University of Padua (Italy), grant “Assegni di Ricerca Junior” 2014 n. CPDR148959, CARIPARO Foundation and the excellence department project of the Italian Ministry of Education, University and research (MIUR) "Centro di Eccellenza per la Salute degli Animali Acquatici - ECCE AQUA" for the support. Moreover, authors gratefully acknowledge the support of the Nanomaterials and Nanotechnologies for Environment Protection and Sustainable Future (NanoEnviCz, LM2015073) and the project LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic. The authors thank the electron microscopy facility of the Biology Department and the Molecular Modelling Section of Padua University. Finally, authors would like to thank Biotecnologie BT S.r.l. for supporting a GC PhD.

ABBREVIATIONS DTT, dithiothreitol; HDL, high-density lipoprotein; HPLC-MS, high-performance liquid chromatography–mass spectrometry HRTEM, high-resolution transmission electron


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microscope; ICP-AES, inductively coupled plasma atomic-emission spectrometry; IONPs, iron oxide nanoparticles; LOQ, limit of quantification; MGF, mascot generic format; MOE, molecular operating environment; OTC, oxytetracycline; PEEP, poly-ethyl ethylene phosphate; PEG, poly ethylene glycol; Q-TOF, quadrupole time of flight; RMSD, root mean square deviation; SAMNs, surface active maghemite nanoparticles; SDS, sodium dodecyl sulfate; TRIS, tris(hydroxymethyl)aminomethane.

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TABLE OF CONTENTS


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Figure 1. Characterization of IONP@OTC complex. Panels A and D show the HR-TEM image of the neat IONPs and panel B the STEM-HAADF image of neat IONP. The Electron dispersive spectroscopy (EDS) mapping of IONP is shown in panels C, E and F with the distribution of oxygen (panel C), iron (panel E) and the elemental overlay (panel F). Panels G and L show the HR-TEM images of IONP@OTC nanoparticles with the STEM-HAADF image (panel H). Electron dispersive spectroscopy (EDS) mapping of IONP@OTC showing the distribution of oxygen (panel I), iron (panel J), nitrogen (panel K), carbon (panel M) and the elemental overlay of Fe,N,C (panel N). Size bars in H, I, J, K, M and N correspond to 10 nm. Panel O shows the optimized DFT structure of the oxytetracycline molecule (RBP86/6-31G*, neutral form, gas phase) with the labelled A, B, C, D ring-chromophores. Panel P shows the comparison of the FT-IR spectrum of neat IONP (upper yellow-trace) and IONP@OTC (lower red-trace). Panel Q shows the powder XRD pattern (Co-Kα radiation) of IONP@OTC nanoparticles. The numbers are the Miller indices for the maghemite phase. The hematite contribution (marked by *) is found below 5 wt %. 169x139mm (300 x 300 DPI)


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Figure 2. Representation of the binding of a dimer of gilthead bream Apo A1 on an iron oxide nanoparticle and gel electrophoresis of proteins bound on iron oxide nanoparticles after incubation in plasma from gilthead bream (Sparus aurata). (A) Apo 1 dimers (a, c) are shown as analytic Connolly’s electrostatic surface charge distribution, calculated using AMBER99 Forcefield. Blue color indicates positive surface charges and red color indicates negative surface charges. The Apo A1 monomer is composed of four antiparallel helixes at N-terminal and two antiparallel helixes at C-terminal domains. Apo 1 dimer bound on iron oxide nanoparticle (b). (B) Electrophoretic profile of proteins released from the surface of iron oxide nanoparticles and IONP@OTC. Proteins bound to iron oxide nanoparticles or IONP@OTC were released by 2 M NH4OH. The most intense bands (at about 60 kDa, marked with an arrow in the gel photograph) were analyzed by LC/MS/MS after tryptic digestion and resulted Apo A1. 82x61mm (300 x 300 DPI)



Figure 3. Analytic Connolly’s electrostatic surface charge distribution of Apo A1 calculated using AMBER99 Forcefield. Blue color indicates positive surface charges and red color indicates negative surface charges. (A) Apo A1 monomers: human (1), gilthead bream (2) and zebrafish (3); (B) Apo A1 trimeric structures: gilthead bream (1) and zebrafish (2); (3) graphical representation of the interaction between Apo A1 trimer and iron oxide nanoparticle (brown). (C) Graphical representation of the “belt mode” tetrameric structure of Apo A1: gilthead bream (1) and zebrafish (2). 82x45mm (300 x 300 DPI)


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Figure 4. Optical micrographs and electron microscopy images of zebrafish intestinal tissue treated with IONP@OTC. (A) Photomicrograph of cross-section of intestine showing intestinal villi in zebrafish treated with 100 mg L-1 IONP@OTC. Each villous is lined by simple columnar epithelium containing absorptive cells with uniform and continuous striated border of microvilli and lamina propria in the core of each villous. No macroscopic aggregates of nanoparticles are detectable. H&E staining. (B, C) Transmission electron microscopic detection of single electron-dense IONP@OTC attached to the surface of microvilli (B, C: arrows). Structures resembling nanoparticles were also observed inside gut cells (B: arrows). (D) Higher magnification of the same region (C) revealing single spherical nanoparticles (arrows) attached to the apical region of microvilli. 82x97mm (300 x 300 DPI)



Figure 5. Optical micrographs and electron microscopy images of zebrafish liver and ovary tissues treated with IONP@OTC. (A) Photomicrograph of cross-section of liver parenchyma in 100 mg L-1 IONP@OTC administered zebrafish, showing the organization in hepatocytes cells. No clusters of nanoparticles are detectable. H&E staining. (B) Transmission electron microscopic detection of hepatocyte cells surrounding a capillary. (C) Higher magnification of the same region (B) reveals single spherical nanoparticles (arrows) inside the capillary. (D) Transmission electron microscopic detection of nanoparticles in the space between the follicular epithelium and the zona radiata of zebrafish ovary. 82x98mm (300 x 300 DPI)


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Stealth Iron Oxide Nanoparticles for Organotropic Drug Targeting

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