Tailor-made PEG coated iron oxide nanoparticles as contrast agents for long lasting magnetic resonance molecular imaging of solid cancers

Journal Pre-proof Tailor-made PEG coated iron oxide nanoparticles as contrast agents for long lasting magnetic resonance molecular imaging of solid cancers Ana Lazaro-Carrillo, Marco Filice, María José Guillén, Rebeca Amaro, Mario Viñambres, Andrea Tabero, Karina Ovejero Paredes, Angeles Villanueva, Pilar Calvo, Maria del Puerto Morales, Marzia Marciello PII:

S0928-4931(19)32186-1

DOI:

https://doi.org/10.1016/j.msec.2019.110262

Reference:

MSC 110262

To appear in:

Materials Science & Engineering C

Received Date: 13 June 2019 Revised Date:

6 September 2019

Accepted Date: 26 September 2019

Please cite this article as: A. Lazaro-Carrillo, M. Filice, Marí.José. Guillén, R. Amaro, M. Viñambres, A. Tabero, K.O. Paredes, A. Villanueva, P. Calvo, M. del Puerto Morales, M. Marciello, Tailor-made PEG coated iron oxide nanoparticles as contrast agents for long lasting magnetic resonance molecular imaging of solid cancers, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/ j.msec.2019.110262. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Tailor-Made PEG Coated Iron Oxide Nanoparticles as Contrast Agents for Long Lasting Magnetic Resonance Molecular Imaging of Solid Cancers

Ana Lazaro-Carrilloa,†, Marco Filice,b,c,d,† María José Guilléne, Rebeca Amaro,f Mario Viñambres,b,f Andrea Tabero,a Karina Ovejero Paredes,b,d Angeles Villanueva,a,g Pilar Calvo,e Maria del Puerto Morales,f Marzia Marciello,b,f*

a

Department of Biology, Universidad Autónoma de Madrid (UAM), Darwin 2,

Cantoblanco, 28049, Madrid, Spain b

Nanobiotechnology for Life Sciences Group, Department of Chemistry in

Pharmaceutical Sciences, Faculty of Pharmacy, Complutense University of Madrid (UCM), Plaza Ramón y Cajal, 28040 Madrid, Spain c

Biomedical Research Networking Center for Respiratory Diseases (CIBERES),

C/Melchor Fernandez-Almagro 3, 28029 Madrid, Spain d

Fundacion Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC),

Melchor Fernandez Almagro, 3, 28029 , Madrid, Spain e

Research Department, PharmaMar S.A., Colmenar Viejo, 28770, Madrid, Spain

f

Department of Energy, Environment and Health, Institute of Materials Science of

Madrid, ICMM-CSIC, Sor Juana Inés de la Cruz 3, Cantoblanco, 28049, Madrid, Spain g

Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia),

Faraday 9, Campus Universitario de Cantoblanco, 28049, Madrid, Spain †

Both authors contributed equally to this work

*

Corresponding author: [email protected]

Authors declare no conflict of interest related to this study.

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Keywords (2-6 keywords): pegylated iron oxide nanoparticles, cancer diagnosis, endocytic mechanism, intratumoral injection, contrast agent, magnetic resonance molecular imaging.

Abstract Magnetic resonance imaging (MRI) is the most powerful technique for non-invasive diagnosis of human diseases and disorders. Properly designed contrast agents can be accumulated in the damaged zone and be internalized by cells, becoming interesting cellular MRI probes for disease tracking and monitoring. However, this approach is sometimes limited by the relaxation rates of contrast agents currently in clinical use, which show neither optimal pharmacokinetic parameters nor toxicity. In this work, a suitable contrast agent candidate, based on iron oxide nanoparticles (IONPs) coated with polyethyleneglycol, was finely designed, prepared and fully characterized under a physical, chemical and biological point of view. To stand out the real potential of our study, all the experiments were performed in comparison with Ferumoxytol, a FDA approved IONPs. IONPs with a core size of 15 nm and coated with polyethyleneglycol of 5 kDa (OD15P5) resulted the best ones, being able to be uptaken by both tumoral cells and macrophages and showing no toxicity for in vitro and in vivo experiments. In vitro and in vivo MRI results for OD15-P5 showed r2 relaxivity values higher than Ferumoxitol. Furthermore, the injected OD15-P5 were completely retained at the tumor site for up to 24 h showing high potential as MRI contrast agents for real time longlasting monitoring of the tumor evolution. 1. Introduction

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Magnetic resonance imaging (MRI) is a powerful, non-invasive, clinical diagnostic tool that provides high-resolution images without the limitation of tissue depth [1, 2]. More than one third of clinical MRI applications rely on the administration of contrast agents [3]. Those should be able to accumulate in a specific area providing an image of the disease but also should be uptaken by cells becoming interesting cellular MRI probes for tracking and monitoring, for example, transplanted stem cells (promising tool for the treatment of various human diseases and disorders)[4] and other human cells such as immune cells associated to different diseases [5-7]. Different MRI contrast agents have been approved already for clinical application. However, their clearance is very fast and their tissue specificity is low being rapidly eliminated from the body. As a consequence, they could promote a weak signal enhancement finally resulting in their limited use for oncological imaging [8]. In this sense, long lasting accumulation of contrast agents in solid tumors could aid in precise tumor excision and would enable postsurgical followup imaging in order to assess the completeness of mass removal without further injections of contrast agent. Therefore, the design and development of more efficient contrast agents for MRI molecular imaging possessing high and lasting MRI signal intensity, good pharmacokinetic properties, adequate safety profile for the patient and finally avoiding repeated administration is a still unsolved challenge [9-12]. Because of their unique physicochemical properties at molecular and cellular levels, inorganic nanoparticles (NPs) and in particular, iron oxide nanoparticles (IONPs), are especially interesting for in vivo applications due to their high biocompatibility and unique magnetic properties [13-16]. However their surface functionalization is mainly responsible for reducing the magnetic signal and determine the biodistribution of IONPs [17].

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Suitable IONPs can be designed not only for diagnosis [13, 18-21] but also for drug delivery [22] and/or as therapeutic agents in magnetic hyperthermia [22, 23]. The remarkable feature of these nanomaterials is their ability to act as theranostic agents performing diagnosis and treatment at the same time. Focusing attention on their contrast properties in MRI, IONPs can be able to substitute the presently used gadolinium-based contrast agents that have shown low blood lifetime and low proton relaxation efficiency. These drawbacks have resulted in higher administration doses rising up human and environmental toxicity [4, 20-22]. For example, Kanda et al. have reported brain abnormalities in patients with previous administration of gadoliniumbased contrast material [24, 25]. To ensure safe and efficient clinical development of IONPs, clear information about formulation design, toxicity and biological NPs behavior are crucial requirements that must be deeply characterized. IONPs have been already approved for biomedical applications by the U.S. Food and Drug Administration (FDA). For example, Feridex® (ferumoxides) is the dextrancoated IONPs approved as an imaging contrast agent for the detection of liver lesions. In 2009, FDA approved Ferumoxytol (trade name: Feraheme™) as iron replacement therapy for iron deficiency anemia in adult patients with chronic kidney disease [26]. Ferumoxytol (FMX) has been used off-label as an MRI contrast agent and as a predictive tool of NP accumulation and thus therapeutic response in both a preclinical [27] and a pilot clinical study [28] and will be used in this work for comparison. Recently, IONPs synthesized by thermal decomposition method in organic media with a uniform core size of 15 nm have shown to be interesting candidates in biomedicine for their crystalline and uniform structure, improved saturation magnetization values and their high therapeutic efficiency of in vivo magnetic hyperthermia [29, 30]. For a systemic intravenous administration, an appropriate polymeric coating is required to

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improve their biocompatibility and blood circulation time. To this aim, polyethylene glycol (PEG) is a good candidate because of its hydrophilicity, biocompatibility and capacity to enhance NPs blood half-time [31, 32]. PEG coated IONPs (with a core size of 15 nm) have shown extremely large heating efficiency under an alternating magnetic field which can lead to an administration dose reduction [33]. Here, we have studied the potential application of tailor-made PEG coated IONPs in terms of in vitro and vivo safety and tested their properties as MRI contrast agent. More in details, IONPs with core size of 15 nm were synthesized and coated with PEG with different molecular weights. Polymer coating was covalently anchored to NPs surface in order to prevent their aggregation in physiological conditions and promote their biocompatibility. NPs with a thin coating able to minimize the hydrodynamic size but at the same time thick enough to prevent aggregation were prepared. A detailed characterization of their physicochemical properties as well as their in vitro and in vivo biological behavior and toxicity has been carried out. Finally, the PEG-coated IONPs showing the highest r2 relaxivity values have been assessed as contrast agent promoting magnetic resonance molecular imaging in mice bearing xenograft human breast cancer obtained by inoculation of MDA-MB-231 cancer cells. To carry out a more complete evaluation of the real potential of our study, all the experiments have been performed by comparison with commercially available FMX.

2. Materials and Methods 2.1. Materials All chemicals and solvents were purchased from Sigma-Aldrich and they have been used as received. FMX (carboxymethyl-dextran coated IONPs) was commercially available from AMAG Pharmaceuticals (Waltham, Massachusetts, USA). All reagents

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for cell cultures were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and were sterilized by filtration (0.22 µm pore size, Merck; Darmstadt, Germany).

2.2. Iron oxide nanoparticles synthesis IONPs were synthesized by solvothermal synthesis in organic medium using iron oleate as precursor, as described in reference 28. The obtained hydrophobic NPs (oleic acid coated NPs, O15) were transferred into water by ligand exchange oleic aciddimercaptosuccinic acid (DMSA) reaction (OD15) [30].

2.3. Iron oxide nanoparticles surface modification DMSA coated NPs (OD15) were modified with polyethylene glycol (PEG) by formation of amide bond between carboxylic and amine groups of DMSA and PEG respectively. In more details methoxypolyethylene glycol amine, (PEG) with two different molecular chain lengths (5 kDa and 20 kDa) were used. Carboxylic groups of DMSA coating NPs were activated adding N-hydroxysuccinimide (NHS) 100 mM and 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) 20 mM at pH 5 to the aqueous suspension of OD15 (at 2 mgFe ml-1). After 1.5 h of activation in mild stirring, the suspension was centrifuged at 4500 rpm for 10 min. The supernatant was removed and an aqueous solution of PEG was added to precipitated NPs in 2 mol PEG: 1 mol DMSA proportion obtaining a final suspension 1 mgFe ml-1 and the final pH was quickly adjusted to 8. This suspension was gently stirred during 15 h. After that, it was centrifuged at 4500 rpm for 15 min and washed with water three times. Then, the PEG coated NPs were dialyzed by using dialysis membranes with a cut-off of 50 kDa and 100 kDa to eliminate the excess of polymer (5 kDa and 20 kDa respectively).

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2.4. Nanoparticles characterization Particle size and shape were studied by transmission electron microscopy (TEM) using a 200 keV JEOL-2000FXII microscope. TEM samples were prepared by placing one drop of a dilute suspension of magnetite nanocrystals in water on a carbon coated copper grid and allowing the solvent to evaporate slowly at room temperature. The mean particle size (DTEM) and distribution were evaluated by measuring at least 200 particles and fitting the data to a log normal distribution. For the highest resolution images, a 200 keV Philips Tecnai 20 microscope was used. Colloidal properties of the samples were studied in a Zetasizer Nano ZS TM, from Malvern Instruments. The hydrodynamic size of the particles in suspension was measured by Dynamic Light Scattering (DLS) diluting the sample in ultrapure water and the electrophoretic mobility was measured as a function of pH at 25 °C, using 10-2 M KNO3 as electrolyte and HNO3 and KOH to change the pH of the suspensions. Hydrodynamic size was calculated by DLS (in intensity). Functionalization of NP surface was checked by Fourier transform infrared measurements (FT-IR) carried out in a Nicolet 20SXC FT-IR spectrometer. Samples were dispersed in KBr at 2 wt% and pressed in a pellet. The IR spectra were registered between 4000 and 400 cm-1. Thermogravimetric (TGA) and differential thermal analysis (DTA) analyses of the magnetite powders were carried out in a Seiko TG/ATD 320 U, SSC 5200. The analysis was performed at room temperature up to 900 °C at a heating rate of 10 °C min−1 in an air flow.

2.5. MRI relaxivity of IONPs

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To evaluate the in vitro efficiency of the PEG coated IONPs as contrast agents, relaxation time measurements were carried out in a MINISPEC MQ60 (Bruker) at 37 °C and a magnetic field of 1.5 T. The results were compared with relaxation times of FMX. The samples were prepared in agar solution (2% w/v) at different concentrations (mM). The relaxivities values (r1 and r2, s−1mM−1) were calculated by the liner fitting of the relaxation rates R1,2 (1/T1,2, s−1) values for each Fe concentration and blank solution, according to equation (1): R1,2 = R1,2 + r1,2 [Fe] (1) Where: R1,2 (s−1) is the relaxation rate in the absence of contrast agent, [Fe] is the contrast agent concentration (mM) and r1,2 (s−1 mM−1) is the relaxivity.

2.6. Cell cultures and nanoparticle incubation Human cell lines MDA-MB-231 (from breast adenocarcinoma, ATCC® HTB-26TM) and PANC-1 (from pancreatic duct epithelioid carcinoma, ATCC® CRL-1469TM) and murine cell line RAW 264.7 (Abelson murine leukemia virus transformed macrophages, ATCC® TIB-71TM) were selected due to their distinct cancer phenotypes and endocytic rates. Cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) with 50 units ml-1 penicillin, 50 µg ml-1 streptomycin and supplemented with 10% fetal bovine serum. Cell cultures were maintained in an incubator (Steri-Cult 200, Hucoa-Erloss; Madrid, Spain) with 5% CO2 atmosphere at 37 °C. Depending on the experiment, cells were seeded in 24-well plates (Corning Inc.; Corning, NY, USA) with or without sterile 10 mm side square glass coverslips (Menzel-Gläser; Brunswich,

8

Germany) placed on each well. The coverslips were sterilized with ethanol and a Bunsen burner. All the work was done in a sterile vertical laminar flow class II biological safety cabinet (MSC-AdvantageTM, Thermo Fisher Scientific). Treatments with IONPs were carried out three days after seeding, when the cells were in exponential growth phase, with approximately 60% confluence. The different IONPs stocks were dispersed by sonication for 5 min in a 50 kHz sonicator bath (Bath Ultrasonic QS3, Scientific Laboratory Supplies; Cardiff, UK) and they were then resuspended in cell culture media at a final concentration of 0.2 mgFe ml-1. The mixture was sonicated for 1 min and immediately offered to cells for 3 h or 24 h.

2.6.1. Quantification of IONPs cell uptake The IONPs uptakes were repeated in the three cell lines (RAW 264.7, MDA-MB-231 and PANC-1) applying the conditions described in the previous paragraph 2.6. After 3 h (for RAW 264.7) or 24 h (for MDA-MB-231 and PANC-1) post incubation, the growth medium containing excess IONPs was aspirated. The cells were washed twice with PBS 1x and they were detached from the wells using trypsin (PANC-1), TrypLE Express Enzime 1x (MDA-MB-231) or by scrapering (RAW 264.7). Cells were then centrifuged at 4ºC, 1300 rpm, 7min, the pellet was resuspend in PBS 1x and cells were counted. After that, cells were centrifuged again and the pellet was resuspend in 50 µl of distilled water. Finally, the digestion of the achieved sample was carried out by mixing 75 µl of diH2O, 50 µl of the cell containing sample, 175 µl of HNO3 and 125 µl of H2O2. The mixture was left at 60°C overnight up reaching its complete clarification. After that, being its value directly correlated to iron ions concentration, the longitudinal relaxation time (T1) of the sample was measured in the relaxometer. The iron concentration was extrapolated using a calibration curve (R2 = 0,9985) previously

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obtained by analyzing the T1 of different concentrations of commercial FeCl3*6H2O salts dissolved in the same digesting mixture used for the sample. Data correspond to mean ± SD values from at least three different experiments.

2.6.2. MTT cytotoxicity assay Cell viability was assessed by MTT colorimetric assay 24 h after IONPs treatments. Cells were incubated for 3 h with a 50 mg ml-1 solution of dimethylthiazolyl-diphenyltetrazolium bromide (MTT, Merck) in culture medium. Then, reduced formazan was extracted with 500 ml DMSO and absorbance was measured at 542 nm in a SpectraFluor spectrophotometer (Tecan Group Ltd; Männedorf, Switzerland). Cell survival fraction was expressed as the percentage of absorption of treated cells in comparison with that of control cells. Data correspond to mean ± SD values from at least six different experiments.

2.6.3. Prussian blue staining Cells seeded onto coverslips were incubated with the different formulations, washed three times with DMEM and fixed in ice-cold methanol. After 5 min, samples were stained with 2% hydrochloric acid and 2% potassium ferrocyanide trihydrate for 15 min. The preparations were then, washed with distilled water and counterstained with 0.5% neutral red for 2 min. Once dried, the preparations were mounted on microscope slides (Menzel-Gläser) with DePex (Serva; Heidelberg, Germany). Images of samples were acquired with an Olympus BX61 microscope equipped with an Olympus DP50 digital camera (Olympus; Center Valley, PA, USA), and processed using Adobe Photoshop 7.0 software (Adobe Systems; San José, CA, USA).

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2.6.4. Transmission electron microscopy of cell sections Cells seeded onto coverslips were incubated with the different IONPs formulations as previously described, washed three times with DMEM and treated with a mixture of 2.5% glutaraldehyde (EM grade,) and 1% tannic acid (TAAB Laboratories Equipment Ltd., UK) in HEPES 0.4 M pH 7.2 for 1 h at room temperature. The cell monolayer on the coverslips was then washed with HEPES, post-fixed with 1% osmium tetroxide (TAAB Laboratories Equipment Ltd.) in H2O, washed with distilled water, treated with 1% aqueous uranyl acetate (Electron Microscopy Sciences; Hatfield, PA, USA), dehydrated with growing quantities of ethanol (Merck) and embedded in epoxy resin 812 (TAAB Laboratories Equipment Ltd.). The samples in BEEM® embedding capsules (Ted Pella Inc.; Redding, CA, USA) were polymerized for 2 days at 60 ºC. Resin was detached from the coverslips by successive immersions in liquid nitrogen and hot water. Ultrathin 60-70 nm thick sections were obtained with a Leica EM UC6 ultramicrotome (Leica Microsystems; Wetzlar, Germany), transferred to 200 mesh Nickel EM grids (Gilder Grids Ltd.; Lincolnshire, UK) and stained with 2% aqueous uranyl acetate and lead citrate (Electron Microscopy Science). Sections were visualized on a JEOL JEM1011 electron microscope (operating at 100Kv) (JEOL Ltd.; Tokyo, Japan) with a Gatan ES1000W Erlanghsen CCD camera (Roper Technologies Inc; Lakewood Ranch, FL, USA).

2.6.5. Statistical analysis Statistical significance for surviving-fraction data obtained from the conducted MTT assays was obtained using one-way ANOVA and Dunnett´s post-test (all groups versus control) analyzed by GrapPad Prism 5 software (La Jolla, CA, USA). Statistically

11

significant differences were labelled as ‘*’ when P < 0.05, ‘**’ when P < 0.01 and ‘***’ when P < 0.001.

2.7. In vivo acute toxicity experiments Maximum tolerated dose (MTD) or maximum tolerated multiple dose (MTMD) was evaluated in CD-1 mice after single or multiple intravenous administration, respectively. CD-1 male mice were used for OD15 toxicity studies and CD-1 female mice were used for OD15-P5 and OD15-P20 toxicity studies. The Maximum Tolerated Dose (MTD) or the Maximum Tolerated Multiple Dose (MTMD) was defined as the dose level with no mortality recorded after a single or multiple bolus administration of the IONPs. After the administrations, animals were observed for clinical signs at fixed intervals, up to 14 days after dosing. Mortality was recorded daily.

2.7.1. Animals Male or female CD-1 mice ranging in weight from 16 to 25 g have been purchased from Harlan (Italy). Animals were housed in individually ventilated cages (Sealsafe® Plus, Techniplast S.P.A.), 5 mice per cage, on a 12-hour light-dark cycle at 21-23 ºC and 4060% humidity. Mice were allowed free access to irradiated standard rodent diet (Tecklad 2914C) and sterilized water. Animals have been acclimatized for five days prior to being individually tattoo-identified. Animal protocols were reviewed and approved according to regional Institutional Animal Care and Use Committees.

2.7.2. Experimental Design

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The tested preparations at different concentrations have been administered intravenously, in the lateral vein of the tail. A graduated 1 ml syringe and a 26G (12 mm) needle have been used. The volume administered was adjusted to the body weight recorded just before administration. The administration volume was 10 ml/kg. Animals were randomly allocated to dose groups and received single or multiple intravenous administration of the formulation. After the administration, animals were observed for clinical signs at fixed intervals, up to 14 days after dosing. Mortality was recorded daily. Body Weight Each animal was weighed on Day 0, every 2-3 days during the observation period and before being sacrificed. Mortality check Carried out at least once a day during the assay. Any mouse showing signs of extreme weakness or toxicity, or in a moribund state, was sacrificed. Clinical observations During the administration day and the observation period, the mice were monitored at least once a day. Any clinical response was being carefully noted. The observations included changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, central nervous and autonomic nervous systems, somatomotor activity and behaviour.

2.8. In vivo assessment of contrast agent potential for MRI of IONPs 2.8.1. Breast cancer animal model Female NOD-SCID IL2 mice, 8 weeks old, were bred and housed in specific facilities (pathogen-free for mice) at the CNIC. All animal experiments were carried out after

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previous approval by the ethics and animal welfare committee at CNIC and were in agreement with the Spanish Legislation and UE Directive 2010/63/EU.

2.8.2. Tumor xenograft mice models To generate the tumor model in mice, we have used a human breast cell line (MDAMB-231) cultured in DMEM+10% Newborn calf serum. Female NOD-SCID IL2 mice, 8 weeks old, were bred and housed under pathogen-free conditions in the animal facilities at CNIC. Prior to injection, tumor cells were trypsin detached, washed twice, and resuspended in PBS to a final concentration of 106cells/13 µL. The cell suspension was then mixed with 5-µL growth factor–reduced Matrigel (BD Biocoat) and 2-µL trypan blue solution (Sigma Aldrich) and maintained on ice until injection. Mice were anesthetized with 5% Isofluorane (Abbott), laid on their backs, and injected with 20-µL cell suspension in Matrigel directly in the fourth mammary fad pad through the nipple with a Hamilton syringe. Tumor growth was monitored weekly using digital calipers, and tumor volume was calculated according to the formula: L × W2/2 = mm3. Imaging studies were performed after 5 weeks after implant when tumor masses reached ∼1 cm3 volume approx.

2.8.3. In vivo MRI experiments In vivo MRI in mice was performed with an Agilent/Varian scanner (Agilent; Santa Clara, CA, USA) equipped with a DD2 console and an active-shielded 205/120 gradient insert coil with 130 mT/m maximum gradient strength and a combination of a volume coil and a 2-channel phased-array (Rapid Biomedical GmbH, Rimpar, Germany). Tumor bearing mice (n=3) were anesthetized with 2% isoflurane and oxygen, and positioned on a mouse bed with constant monitoring of respiratory cycle). Ophthalmic

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gel was placed in their eyes to prevent retinal drying. Baseline images were acquired before intratumoral administration of the probes (100 µl at 1 mgFe ml-1 for both OD15P5 and FMX). MRI data were acquired just after contrast agent injection, 7 and 24 h post administration. Axial images were acquired in free-breathing animals, using a gradient echo 3D sequence with echo (TE)/repetition time (TR): 1.65 ms/3 ms; FOV: 27.8 x 30.33 x 26.44 mm; averages: 5; flip angle of 20º and a matrix: 256x256x256. Qualitative Image analyses were performed using Osirix software (Pixmeo, Switzerland).

2.8.4. Histolological analysis of tumor At 24 h after administration of IONPs (OD15-P5 or FMX), mice were sacrificed and the tumor masses were harvested and fixed with 10% formalin for 24 h. Then, the tissues were embedded in paraffin, until sectioning. Tumor sections were stained with Hematoxilin & Eosin, Perl´s Prussian Blue and CD31 immunostaining. Images were processed and digitalised with NanoZoomer Digital Pathology 2.5.19 acquisition software from Hamamatsu.

3. Results and discussion 3.1. Synthesis and characterization of PEG coated nanoparticles Monodispersed hydrophobic IONPs with a core size of 15 nm were synthesized by organic route and transferred to aqueous medium by oleic acid-DMSA ligand exchange reaction (OD15) [30] (Fig 1). With the aim to improve their colloidal stability and biocompatibility, monoamine functionalized PEG was used to coat the OD15 surface. To evaluate whether the polymer chain length could produce a different biological behavior in vitro and/or in vivo application, two different molecular weights (5 kDa and

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20 kDa) of selected PEG were chosen. In more details, PEG was covalently grafted to OD15 surface by EDC chemistry after previous activation of carboxyl groups present on the IONPs surface due to the presence of DMSA. As a consequence of this modification strategy, the polymer grafting density was limited by PEG chain entropy that led to a random coil polymer conformation [34]. The presence of random coil conformation of PEG surrounding OD15 was confirmed by the hydrodynamic size measurement that was around 100 nm (Table 1). In fact, in case of stretched structure, higher size would have been obtained (Fig. S1, Electronic Supplementary Information). For biomedical applications, the time dependent colloidal stability of a given nanoparticle is a key parameter to be evaluated. Thus, in order to assess the water-stability of PEG-coated IONPs, the hydrodynamic size of the best candidate for in vivo application was analyzed 7 and 30 days after its synthesis (Fig S2, Electronic Supplementary Information). The final achieved value (101nm) was the same that has been obtained at the beginning, confirming the optimal colloidal stability of our OD15-P5 IONPs (Fig S2, Electronic Supplementary Information). The presence of PEG on OD15 surface was confirmed by ζ−potential values at different pH (Fig. S3, Electronic Supplementary Information). In fact, the surface potential of OD15 NPs clearly increased from -26.2 mV to -19.4 mV and -14.9 mV at physiological pH in the presence of both PEGs, indicating the reduction of free carboxylic groups and thus confirming the successful PEGylation reaction (Table 1). These results were confirmed also by TGA and IR analysis (Fig. S4 and S5, respectively. Electronic Supplementary Information). The same physical characterization was also extended to commercially available FMX NPs, showing a quite similar surface potential at pH 7 (21.6 mV) but a more reduced hydrodynamic size (30 nm) (Fig. 1 and Table 1).

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3.2. Cell culture 3.2.1. Cytotoxic Assays Before assessing PEG coated OD15-NPs as bioimaging tool in vivo, in vitro biocompatibility was evaluated and compared with commercial FMX in different cell lines:

breast

human

adenocarcinoma

(MDA-MB-231),

pancreatic

human

adenocarcinoma (PANC-1) and mouse macrophage (RAW 264.7). MTT assay was performed 24 h after incubation with the different nanoformulations (Fig. 2). This technique and protocol have been shown to be the most suitable in analysis of biocompatibility [35]. In fact, considering that toxicity of particles depends on many factors (including surface area, size and shape, coating, surface charge, purity, structural distortion, and bioavailability), a new nanomaterial requires an in vitro biocompatibility assessment using different cell types before to be translated to in vivo applications [36]. As general consideration, all the tested nanoformulations did not show any significant cytotoxicity for the assessed concentration (0.2 mgFe ml-1) and in the three tested cell lines (Fig. 2). By one hand, the OD15 alone showed a slight toxicity towards both tumor cell lines (MDA-MB-231 and PANC-1). On the other hand, the presence of PEG on OD15 surface increased their biocompatibility, showing a cell viability similar to the control, both for OD15-P20 and OD15-P5. Similar results in cellular viability were achieved using FMX (Fig. 2). Experiments with macrophages were carried out at 3 h incubation to avoid a high cytotoxic effect triggered by longest incubation time. This outcome is easily explained by the increased internalization ability in macrophages, which would induce higher cytotoxicity in this cell line [26, 37-42]. For all the tested NPs, no toxicity toward macrophages was appreciated.

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3.2.2. Nanoparticles uptake in different established cell lines To demonstrate the impact of the polymer coating on the biological behavior of these NPs, cell uptake experiments were carried out in the three cell lines used in this study. Prussian blue staining (specific for iron detection) showed always a perinuclear distribution (out of nuclei) which seems to be related to a lysosomal pattern [43-45]. Differently from FMX results, a 100% cell labelling was detected for PEG coated NPs (Fig. 3). In fact, OD15-P5 exhibited a proper balance between internalization efficiency and aggregation grade with respect to OD15-P20, which formed bigger aggregates inside the cell. For this reason, OD15-P5 NPs were selected as the most suitable candidates for further studies. Analysing the results in more details, both MDA-MB-231 and PANC-1 cell lines showed a great internalization of OD15-P5 and OD15-P20 IONPs in both cases. In the case of FMX, the uptake in MDA-MB-231 and PANC-1 cells was almost negligible (Fig.3). Conversely, in comparison with both tumour cell lines, a much higher internalization ability of OD15-P5 and OD15-P20 was detected when using RAW 264.7 cells whereas the FMX uptake, even if higher than tumoral cell lines, was very limited (Fig. 3). In a second experimental set, the IONPs uptake was also quantified in order to confirm the trend observed with the Prussian blue mediated qualitative assessment of IONPs internalization. Toward this scope, the uptake experiments described above have been repeated in the same conditions and using OD15-P5 as representative sample in comparison with commercial FMX. After finishing the internalization experiments, the cells have been thoroughly washed, harvested and digested with strong oxidizing agents in order to dissolve the internalized IONPs. The iron cations proceeding from the dissolved IONPs have been titrated by analysing the longitudinal relaxation time (T1) of

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that digestion solution and extrapolating the Fe concentration values using a specific calibration curve. The achieved results were in agreement with the qualitative experiments (Fig. S6, Electronic Supplementary Information). In fact, in the case of OD15-P5 by RAW 264.7 cell line the relaxometry-mediated titration retrieved an amount of 226 pgFe cell-1 whereas in the case of both tumoral lines the iron concentration was 10 pgFe cell-1. In the case of FMX, the uptake amount was not detectable in both tumoral lines while it reached a value of 10 pgFe cell-1 in the case of RAW 264.7 internalization (Fig. S6, Electronic Supplementary Information). Our findings are in agreement with those of other authors, who showed that macrophages lines internalized higher levels of NPs when compared to other cancer lines [34, 35] excepting in the case of FMX. In fact, Cao et al. showed that the tumor cell line (MDA-MB-435) is not able to internalize FMX [46] while Pham et al. have described that RAW 264.7 macrophages are able to accumulate iron at concentrations as high as 300 µg/106 cells after 12 h of incubation with SPION at 25 µgFe ml-1 [47]. TEM visualization comparing uptake degree of OD15-P5 IONPs in MDA-MB-231 and RAW264.7 cell lines confirmed higher internalization ability in macrophages (Fig. 4). Thus, macrophages displayed very high NPs internalization at 24 h, both by number and aggregate size, which may impair proper cellular transport and function. Incubation for 3h was enough to label 100% macrophages with a suitable amount, unlike MDA-MB231 cells behavior. Considering that the TEM analysis has been proposed as a very appropriate methodological technique to distinguish between different forms of cell death [48], these studies enabled us also to confirm the biocompatibility of OD15-P5 NP. In fact, cytoplasmic membrane was not altered (without blebs or discontinuances), nuclei showed normal condensation and vacuolization in the cytoplasm (the most usual alterations induced by cytotoxic particles) was not observed (Fig. 4).

19

In-depth TEM studies allowed us to determine also the specific endocytic mechanism in the uptake of OD15-P5. Also in this case, it should be noted that TEM analysis is considered within the most suitable and trusty techniques to identify the specific endocytic mechanism of IONPs [49-51]. It is well known that different kinds of endocytosis pathways are the major route involved in the entrance mechanism of NPs into cells. The specific endocytic route is determined by a set of physicochemical properties of particles (e.g. size, shape and surface chemistry of the NPs) and cell types [52-54]. In addition, many NPs tend to an agglomeration state under physiological conditions, which can affect their mechanism of internalization into cells [55]. Based on the TEM micrographs, OD15-P5 IONPs were internalized in MDA-MB-231 cells by two different mechanisms, according to aggregates size: i) caveolae-dependent endocytosis, to internalize groups of NPs smaller than around 200 nm and ii) macropinocytosis, to internalize the largest aggregates (Fig. 5a and b).There are evidences that the caveolae-dependent pathway can bypass lysosomes, resulting in a direct deliver to the endoplasmic reticulum or the Golgi apparatus. For this reason, this route is believed to be beneficial for nanomaterial drug delivery [53, 56]. For example, a similar caveolae-mediated endocytosis uptake pathway has been already described for SPIONs and silica-coated iron oxide NPs (SCIONs) in HeLa cells [57]. On the other hand, there are still some controversies regarding the precise mechanism of NP entry in relation to its size. In fact, some studies showed that the uptake of particle smaller than 200 nm in diameter was mediated by clathrin-mediated endocytosis, and when their size was increased, uptake shifted to caveolae-mediated internalization. On the contrary, other studies have described caveolae-dependent internalization of 50-80

20

nm-diameter NPs, while NPs of 120 nm diameter were internalized by clathrin-coated pits [58, 59]. Macrophages

engulfed

OD15-P5

primarily

by

phagocytosis

(pseudopodial

envelopment, Fig. 5c) and, in a significant minor extent, by caveolae-mediated endocytosis (Fig. 5d). These predominant mechanisms observed in RAW 264.7 cells were previously described in micellar NPs [60]. Considering the overall in vitro results, the OD15-P5 NPs showed the best properties to be selected as most valuable candidate for the further in vivo assessment. Once selected the best IONPs candidate, we decided to better characterize the in vitro toxicity of the OD15-P5 IONPs in comparison with commercial FMX before to assess their application for in vivo experiments. Toward this scope, the IONPs dose-dependent MTT cell viability assay was performed in the conditions previously described. The achieved results indicate that, when used in higher concentration than those previously assessed (0.2-1 mgFe ml-1), the OD15-P5 IONPs expressed a certain toxicity degree mainly against tumoral cells (Fig. S7a, Electronic Supplementary Information). For example, when used at 1 mgFe ml-1 concentration, these nanoparticles reduced the cell viability up to 70% and 60% roughly, of MDA-MB-231 and PANC-1 respectively (Fig. S7a, Electronic Supplementary Information). In the case of macrophages, only a slight cell viability reduction was observed (≈ 10%) at the same concentration (Fig. S7a, Electronic Supplementary Information). On the other hand, at the same concentrations, the FMX IONPs showed no toxicity toward both tumoral cells while promoted a slight cell viability reduction in the case of macrophages (≈ 10%) (Fig. S7b, Electronic Supplementary Information).

3.3. MTD and MTMD experiments

21

After having proved the NPs viability in vitro, the in vivo acute toxicity was also evaluated. The Maximum Tolerated Dose (MTD) -defined as the highest dose of a chemical that does not cause toxicity in mice or rats- is an important parameter to be considered in diseases like cancer [61]. Indeed, it is a valuable parameter that must be taken in consideration in animal models to study in vivo toxicological effects and subsequently in humans for Phase I cancer clinical trials. Therefore, MTD values and Maximum Tolerated Multiple Dose (MTMD) of PEG coated NPs were evaluated in mice in order to determine a maximum dose that can be administrated without obtaining any toxic effect in vivo. The iron concentration used in the toxicity experiments ranged from 10 up to 4.3 mgFe ml-1 depending from the obtained NPs batch. For MTD experiments, the formulations were administered intravenously in a single injection to CD-1 mice at a pre-established dose (10 mgFe ml-1 for OD15 study, 4.3 mgFe ml-1 for OD15-P5 study and 4.5 mgFe ml-1 for OD15-P20 study) (Table S1, Supporting Information). No mortality was registered at the dose and schedule assayed but due to the clinical signs registered (motor and respiratory activity decrease) within the animals after being administered, it can be concluded that these formulations have to be administered in a very slow bolus and immediately after being sonicated. In MTMD experiments with OD15, OD15-P5 and OD15-P20, the formulations were administered intravenously in a multiple schedule (five daily administrations) to CD-1 mice at a pre-established dose (5 mgFe ml-1 for OD15 study, 4.8 mgFe ml-1 for OD15-P5 study and 4.5 mgFe ml-1 for OD15-P20 study). No mortality was registered in OD15 and OD15-P5 at the assayed dose and schedule (Table S1, Electronic Supporting Information). However, the administration of OD15-P20 formulation in this experiment, resulted in mortality. In fact, due to poor conditions (back limbs paralysis and curved

22

spine), one animal was sacrificed on day 5. A necropsy at macroscopic level was performed resulting in a damaged liver with a red-dark color, probably due to particles accumulation (Fig. S8, Electronic Supporting Information). For this reason, OD15-P5, that also shown best cell uptake results in vitro, was chosen as best candidate to be used in for MRI experiments in vitro and in vivo and compared with FDA-approved IONPs FMX.

3.4. In vitro relaxation measurements To evaluate the OD15-P5 impact on the potential application as contrast agent in MRI, longitudinal (R1) and transverse (R2) relaxation rates at 1.5 T were measured and compared with those of the commercial FMX NPs. In order to compare the results reported in the Table 2, r1 and r2 values of another commercial IONPs (Endorem®), showing similar core size and higher hydrodynamic size than FMX, already described in literature, were also listed (Table 2). OD15-P5 showed values of r1 comparable with those of the commercial samples while r2 values were much higher. Consequently, also r2/r1 ratio resulted very high (>24), confirming the potential ability of OD15-P5 to be used as T2-contrast in MRI. Furthermore, thanks to high value of SAR showed in previous studies [33], OD15-P5 NPs could be also considered as good candidate for therapeutic magnetic hyperthermia, thus, enabling potential cancer theranosis.

3.5. In vivo assessment as contrast agents for magnetic resonance molecular imaging of tumor In order to demonstrate that OD15-P5 are of high interest as MRI contrast agents because their potential high-sensitivity and long lasting MR imaging abilities in vivo,

23

these NPs were injected in mice tumor xenograft models and compared with the commercial FMX nanoparticles. To this scope, MDA-MB-231 xenograft tumors (∼1 cm3) were grown in immunodeficient mice. The same amount of contrast agents FMX and OD15-P5 was directly injected into the tumor mass. MR images were acquired using a volumetric coil with a gradient echo 3D sequence and at different time points in order to follow the kinetic variation of MRI contrast in various tumor regions and over the time. In fact, thanks to the selected MRI acquisition modality, a slice-by-slice analysis of whole tumor has been carried out, finally enabling us to achieve a global 3D reconstruction of the complete contrast area generated by the nanoparticles injected inside the tumor mass. By this strategy, we have been able to visualize the volume occupied by the injected IONPS through the tumor and assess their lasting abilities by comparing their eventual time-dependent volume decrease (Fig. S9, Electronic Supplementary Information). In the case of FMX, after 7 h post-injection (p.i.), the reconstructed IONPs volume decreased up to the 32% of the initial value, 0.0689 cm-3 vs 0.2153 cm-3 respectively (Fig. S9, Electronic Supplementary Information) and reached almost background level within 24 h (Fig. 6). On the other hand, even after 24 h post-injection, the OD15-P5 contrast agent maintained almost entirely the initial volume, 0.215 cm-3 vs 0.229 cm-3 respectively (Fig. 6 and S9, Electronic Supplementary Information). This evidence indicates optimal pharmacokinetic and cell internalization properties finally resulting in a long lasting molecular imaging enhancement, undoubtedly more useful than the commercial contrast media (Fig. 6b vs 6e). These in vivo results were in agreement with those achieved with in vitro cell uptake analysis (Fig. 3). These promising results were further confirmed by immunohistochemistry analyses of tumor sections in order to identify the presence of IONPs within tumor tissue and clarify the

24

different behavior. In the case of FMX, after 24 h p.i., no clear accumulation of NPs can be further appreciated in the neighboring injection area by Prussian blue staining (Fig. 6f). Conversely, a diffuse distribution of IONPs can be clearly observed mainly within the tumor periphery (Fig. 6f, inset). These tumor peripheries correspond in general with the zones showing the highest neovasculature density. Hence, the presence of NPs indicates that they have escaped from their initial inoculation point being then spread out through blood systemic circulation and accumulated in tumor periphery by EPR effect [62]. CD31 immunostaining - used primarily to demonstrate the presence of endothelial cells in histological tissue and thus the vasculature zone - of the same section analysed by Prussian Blue demonstrated the overlapping of NP clusters within the peripheral blood vessels corresponding with neoangiogenesis zone, finally confirming this ‘washing out’ hypothesis (Fig. S10, Electronic Supplementary Information). On the other hand, in the case of OD15-P5, after 24 h, a clear accumulation of the NPs can be still observed in the initial inoculation zone (Fig. 6c). Differently than FMX, no diffuse distribution of IONPs can be clearly appreciated in the tumor periphery, confirming the good retention of OD15-P5 over time. This behavior is extremely useful for longitudinal imaging studies as well as for therapeutic strategies such as magnetic hyperthermia or anticancer drug prolonged delivery.

4. Conclusions Iron oxide nanoparticles coated with PEG of 5 kDa and with hydrodynamic size around 100 nm have shown in vitro biocompatibility and in vivo safety to be considered suitable candidates for biomedical application. The cell uptake in tumoral cells or in

25

macrophages confirms them as a good tool for cancer treatment (by magnetic hyperthermia and/or drug delivery) as well as for longitudinal imaging studies. Ferumoxytol, FDA-approved iron oxide nanoparticles for intravenous treatment of iron deficiency in patients and showing properties of contrast agent for MRI, has been used as model to compare the nanoparticles studied in this work. In vitro MRI results have shown higher r2 relaxivity values for OD15-P5 NPs with respect to Ferumoxytol. These results were also confirmed in vivo. Indeed, due to the tumor cell uptake, the injected OD15-P5 agent was retained completely at the tumor site for up to 24 h while Ferumoxytol was completely washed away within 24 h. These results suggest that these particles are suitable for longitudinal imaging studies as well as for therapeutic applications thanks to their high value of SAR. Considering the current interest in non-invasive diagnostic assay based on real-time and long-time tracking and monitoring of labelled cells (i.e. tumor associated macrophages), these biocompatible nanoparticles represent a powerful tool to be used for biomedical applications.

Acknowledgements This work has been supported by European Seventh Framework Programme (Multifun project 262943), Spanish Ministry for Economy and Competitiveness (MINECO) (CTQ2016-78454-C2-2-R and MAT2017-88148-R). A.L.-C. acknowledges financial support from UAM (Teaching Assistant contract 20140513-136). The CNIC is supported by MINECO and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.F. would like to thank MINECO for the research grant no. SAF2014-59118-JIN co-funded by Fondo Europeo de Desarrollo Regional (FEDER) and COST Action CA1520: ‘European Network on NMR

26

Relaxometry-EURELAX’. M.F. acknowledges the Comunidad Autonoma de Madrid for research project no. 2017-T1/BIO-4992 (“Atracción de Talento” Action) also cofunded by Universidad Complutense de Madrid. M.M and M.F. are grateful to the Comunidad Autonoma de Madrid and FEDER for the I+D collaborative Programme in Biomedicine NIETO-CM (Project reference B2017-BMD3731). IMDEA Nanociencia acknowledges support from the 'Severo Ochoa' Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686). We gratefully thank Cristina Patiño for technical assistance with electron microscopy samples and the personal responsible of the chemical analysis (FT-IR, TGA) of ICMM.

Authors declare no conflict of interest related to this study.

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Figures

Figure 1 TEM micrographs. TEM micrographs of commercial Ferumoxitol® compared to OD15 Left column: core size distribution (log-normal fit) of Ferumoxitol® and OD15.

30

Figure 2 Cell viability by MTT assay. Biocompatibility was analyzed 24 h after incubation of MDA-MB-231 (blue), PANC-1(red) and RAW 264.7 (green) cells with 0.2 mgFe ml-1 of the different IONPs. Data correspond to mean ± SD values from at least six different values. Statistically significant differences are labelled as ‘*’ when P < 0.05, ‘**’ when P < 0.01 and ‘***’ when P < 0.001, for comparisons between groups using one-way ANOVA and Dunnett´s post test (all groups versus control).

31

Figure 3 Uptake of IONPs into cells visualized by optical microscopy. Prussian blue staining in control MDA-MB-231, PANC-1 and RAW 264.7 cells or incubated with the different IONPs (blue spots) at 0.2 mgFe ml-1 for 3 h (RAW 264.7 cell line) or 24 h (MDA-MB-231 and PANC-1 cell lines). Scale bar 10 µm.

32

Figure 4 Uptake and accumulation of OD15-P5 IONPs into cells visualized by electron transmission microscopy. (A) Analyses of uptake and accumulation kinetics in MDA-MB-231 or RAW 264.7 cells incubated for 1, 3 or 24 h with OD15-P5 IONPs. Scale bar 5 µm. (B) MDA-MB-231 or RAW 264.7 cells incubated for 3 or 24 h with OD15-P5 IONPs. Scale bar 5 µm.

33

Figure 5 Internalization mechanisms of OD15-P5 MNPs in cells visualized by electron transmission microscopy. (a) Macropinocytosis of larger aggregates or (b) Caveolae-mediated endocytosis of smaller aggregates in MDA-MB-231 cells. (c) Phagocytosis most common internalization mechanism and (d) caveolae-mediated endocytosis less often in RAW 264.7 cells. Scale bar 100 µm.

34

Figure 6 Coronal view for T2-weighted gradient echo 3D MRI of the tumor area in a mouse after the injection of IONPs (a, OD15-P5 and d, Ferumoxytol) and 24 h postinjection (b, OD15-P5 and e, Ferumoxytol). Prussian blue staining of tumor section 24 h post-injection (c, OD15-P5 and f, Ferumoxytol).

35

Tables

Table 1. Hydrodynamic size (HS), polydispersity index (PDI) and ζ potential values of the prepared IONPs such as of the commercial nanoparticles Ferumoxitol® (FMX). ζ Pot pH 7

HS Sample

PDI (nm)

(mV)

OD15

60

0.17

-26.2

OD15-P5

115

0.19

-19.4

OD15-P20

109

0.21

-14.9

FMX

30

0.15

-21.6

36

Table 2. Relaxometric values (r1 and r2), core size and hydrodynamic size of OD15-P5 compared with the commercial samples FMX (measured values) and Endorem® (values reported in literature). Core size

Hydrodynamic

Sample

r1 (mM-1s-1)

r2 (mM-1s-1)

r2/r1

(nm)

size (nm)

OD15-P5

15

115

7.8

189.94

24.35

FMX

3.7

30

16.7

81

4.85

3-5

120-180

10.1

120

11.88

Endorem (Guerbet)[63]

37

Table 2. Relaxometric values (r1 and r2), core size and hydrodynamic size of OD15-P5 compared with the commercial samples FMX (measured values) and Endorem® (values reported in literature). Core size

Hydrodynamic

Sample

r1 (mM-1s-1)

r2 (mM-1s-1)

r2/r1

(nm)

size (nm)

OD15-P5

15

115

7.8

189.94

24.35

FMX

3.7

30

16.7

81

4.85

3-5

120-180

10.1

120

11.88

Endorem (Guerbet) [63]

38

Highlights: • • • •

Tailor made Iron oxide nanoparticles (IONPs) coated with polyethylene glycol (PEG) In vitro and in vivo biocompatibility of PEG coated IONPs Suitable contrast agents for real time long-lasting MRI of solid cancers Complete retention of injected PEG coated IONPs at the tumor site for up to 24 h