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Family of Bioactive Heparin-Coated Iron Oxide Nanoparticles with Positive Contrast in Magnetic Resonance Imaging for Specific Biomedical Applications Hugo Groult,† Nicolas Poupard,† Fernando Herranz,‡ Egle Conforto,§ Nicolas Bridiau,† Fréderic Sannier,† Stéphanie Bordenave,† Jean-Marie Piot,† Jesús Ruiz-Cabello,‡ Ingrid Fruitier-Arnaudin,† and Thierry Maugard*,† †

UMR CNRS 7266 LIENSs, Approches Moléculaires Environnement-Santé environnement (AMES), University of La Rochelle, La Rochelle, France ‡ Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain § UMR CNRS 7356 LaSIE, University of La Rochelle, La Rochelle, France S Supporting Information *

ABSTRACT: Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH) are well-known for their anticoagulant properties. There is also currently a growing interest in using LMWH in targeted cancer therapy. In particular, several types inhibit heparanase, a key enzyme overexpressed in the tumor microenvironment that promotes angiogenesis progression and metastasis spreading. Here, we propose iron oxide nanoparticles (HEP-IONP) coated with different heparins of distinct anticoagulant/anti-heparanase activity ratios and suitable for positive contrast in magnetic resonance imaging. As a proof of concept, magnetic resonance angiography (MRA) was conducted in mice up to 3 h after intravenous administration. This new IONP-based positive contrast appropriate for clinic together with the long vascular circulating times can enable innovative theranostic applications if combined with the various bioactivities of the heparins. Indeed, we showed, using advanced in vitro tests, how HEP-IONP anticoagulant or antiheparanase activities were maintained depending on the heparin species used for the coating. Overall, the study allowed presenting an IONP coated with a commercial LMWH (Lovenox) suggested as a theranostic translational probe for MRA diagnostic and treatment of thrombosis, and an antitumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.

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glucosamine residues.6 UFH and its depolymerized derivatives, the low-molecular-weight heparins (LMWH, MWavg < 8 kDa), are better known for their anticoagulant properties, and have been used in clinical practice for years.7 They also generate a rising interest in research to control tumor growth with an activity in angiogenic and metastatic processes.8 Among the underlying mechanisms that have not yet been fully clarified, the strong capacity of heparins to bind growth factors and inhibit heparanase, a hydrolytic enzyme overexpressed in the tumor microenvironment, may be mentioned.9,10 This glycosidase selectively cleaves heparan sulfate (HS) proteoglycans that are found in the extracellular matrix (ECM), and participates in matrix remodeling and vascular basement membrane degradation. This activity controls the release of numerous pro-angiogenic signaling molecules and facilitates tumor cell extravasation.11 Although the application of heparins as anticancer agents has long

INTRODUCTION

It is widely appreciated that nanomedicine is currently one of the best strategies for medical research.1 It allows the vectorization of bioactive compounds and a possible diagnostic function for monitoring disease response to treatments, two major features in the development of improved therapies.2 Among the nanomaterials, iron oxide nanoparticles (IONP) are of particular interest. Indeed, due to their unique magnetic features and high biocompatibility, they have been intensively used in preclinical and clinical studies such as magnetic resonance imaging (MRI), cell sorting, targeted or controlled drug delivery, and hyperthermia treatment against tumors.3,4 One of the key challenges in the development of IONP for biomedical applications is their surface functionalization with biomolecules that ensure either a good colloidal stability and bioacceptability, a therapeutic function, or an in vivo targeting.5 Native unfractionated heparin (UFH) is a complex natural polysaccharide belonging to the glycosaminoglycan family and characterized by long linear chains (MWavg ∼ 12 kDa) made of variably sulfated disaccharide units of 1,4-linked uronic acid and © 2017 American Chemical Society

Received: June 7, 2017 Revised: August 15, 2017 Published: August 29, 2017 3156

DOI: 10.1021/acs.biomac.7b00797 Biomacromolecules 2017, 18, 3156−3167

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Biomacromolecules been limited by its main anticoagulant properties and other side effects, new heparins have recently been generated with discriminated anti-heparanase activity and a strong capacity for the sequestration of selective pro-angiogenic growth factors while showing no/reduced anticoagulant activity.12 Several of these molecules are now undergoing phase III clinical trials as potential antiangiogenic and antimetastatic drug candidates.13 UFH and LMWH are increasingly used in the composition of nanomaterials for applications in tissue engineering, cancer therapy, or pharmacokinetics owing to their unique physicochemical and biological properties along with their easy chemical functionalization. Excellent reviews by Yang et al. and Linhardt et al. summarize the multiple applications of these heparin-based nanosystems.14,15 However, most of them incorporate heparin as a coating or backbone to tailor or improve physicochemical properties of nanocarriers that transport an exogenous drug. Surprisingly, only a few studies have functionalized metallic nanoparticles with heparins considering them as the main therapeutic group,16−18 in particular the newly proposed heparins used as anticancer agents. In addition, the UFH-based tumor-targeting properties sometimes proposed for nanosystems remain unclear in front of the enhanced permeability and retention (EPR) effect.9,19−22 Besides, in many proposed carriers, “classic” tumor-targeting moieties for nanoparticles have been added such as folic acid, antibodies, or arginine-glycineaspartic acid (RGD) peptide.14 Thus, regarding IONP, UFH and LMWH have mainly been proposed as a coating material to improve their colloidal stability,20,23,24 bioacceptability,25,26 and hemocompatibility.27−29 They have also been described as a suitable coating to load bioactive agents on the IONP surface, such as chemotherapeutics,30,31 proteins,32,33 virus,34 or stem cells,35−37 in studies of drug delivery with a T2 negative contrast MRI monitoring. To date, IONP functionalized with heparin derivatives displaying anticancer activity has not been reported yet. Moreover, in all of these approaches, iron oxide cores have been synthesized with the traditional coprecipitation method and UFH or LMWH have then been adsorbed on the surface25,33 otherwise attached to a pre-existing coating either covalently28,32 or electrostatically.27 In addition to the cumbersome multiplestep synthesis, these methods produce nanoparticles of relatively large size.25,27,30 The use of these IONP as T2-negative MRI contrast agents is also continuously debated (mislead diagnosis, blooming effect) compared to the more accurate and favorable T1-positive contrast agents used in clinical practice, with added possibility for angiography.38−40 A review by Shen et al. summarizes the recent progress that succeeded to obtain suitable T1-positive MRI agents from IONP using very small particles of core below 5 nm with lowered magnetization in order to reduce the T2 effect and enhance the T1 contrast power.41 This next generation of IONP-based T1 contrast agents may represent a remarkable alternative to the drawbacks of the gadolinium (Gd)-based mainstream positive MRI contrast agents (nephrotoxicity, Gd deposition, short vascular lifetime)40,42 by bringing several opportunities such as nontoxicity, blood pool capability for longer efficient imaging time, and multifunctionality including targeting, therapy, or multimodal imaging.39,43−45 Many methods have been applied for the preparation of very small IONP; among them, microwave (MW) synthesis got an up-going interest. Indeed, fast and selective MW dielectric heating together with technical simplicity enable the reaction times to be reduced considerably, the yields to be increased, and the reproducibility to be enhanced over other approaches.46

IONP with dextran coating produced by MW technology have lately been proposed as suitable multifunctional T1-MRI contrast agents.38,47 Here, a one-step microwave-assisted method for the rapid synthesis of IONP was adapted to produce heparin functionalized very small IONP (HEP-IONP) that could be used for the first time as T1-positive contrast agents in MRI. The method was applied on three heparins (UFH and two LMWH) displaying different anticoagulant and anti-heparanase activities. The resulting bioactivities of the IONP were carefully investigated to determine whether heparins could act both as a stabilizing surfactant and as a therapeutic functional group. The aim of this study was to develop new theranostic probes with specific bioactivity and, at the same time, a T1-positive magnetic resonance contrast.

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EXPERIMENTAL SECTION

Materials. All reagents, unless otherwise specified, were purchased from Sigma-Aldrich. Porcine mucosal heparin sodium salt (UFH, 163 U·mg−1) was purchased from Interchim. Commercial LMWH: Enoxaparin sodium (Lovenox, 10,000 anti-Xa U·mL−1) was kindly provided by the La Rochelle Hospital (France). Synthesis of the LMWH Named usHEP. The LMWH named usHEP was prepared in our laboratory from UFH (Alpha Aesar, batch 10124103), with a hydrogen peroxide catalyzed radical hydrolysis assisted by ultrasonic waves, as previously described.48 Briefly, UFH (100 mg) was first resuspended in 4 mL of Milli-Q water before addition of a 30% hydrogen peroxide solution (46 μL) to obtain a final H2O2/UFH (w/w) ratio of 0.15 (3.75 mg·mL−1). The reaction mixture was sonicated (3 mm diameter probe type UP50H utrasonicator, Hielscher, Germany) at a pulse of 0.5 s with a frequency of 30 kHz in a Radleys reactor for 5 h at room temperature (RT). The solution was finally dialyzed (cutoff: 1 kDa) and lyophilized to obtain pure usHEP (80 mg, 80% yield). Structural and Quantitative Analysis by Size Exclusion Chromatography. Structural and quantitative analyses of heparins by size exclusion chromatography (SEC) were performed using a LC/MS-ES system from Agilent (1100 LC/MSD Trap VL mass spectrometer) with one TSK-GEL G3000PW and two TSK-GEL G2000PW columns mounted in series. The columns were maintained at 30 °C, and the products were eluted with 0.1 M sodium nitrate (NaNO3) at a flow rate of 0.5 mL·min−1. The products were detected and quantified by differential refractometry using a HP Chemstation software off-line for the processing. Heparin oligosaccharides of different molecular weights purchased from Iduron (Manchester, U.K.) were used for the calibration curve. The number-average molecular weight (Mn), weight-average molecular weight (Mw), degree of polymerization (DP), and polydispersity index (PIheparins) were calculated using the following equations49 M n = (∑ Ni × Mi)/∑ Ni ; M w = (∑ Ni × Mi 2)/(∑ Ni × Mi);

DP = M n /M 0 ;

PI = M w /M n where Ni was the number of moles of polymer species, Mi the molecular weight of polymer species, and M0 the molecular weight of monomer unit. Quantification of the Degree of Sulfation of Heparins. The degree of sulfation (DS) on the sugar backbone was monitored using (7-aminophenothiazin-3-ylidene)-dimethylazanium chloride (Azure A), which binds the sulfated groups to form a colored complex.50 In a 96-well plate, 20 μL of a serial dilution (0 to 30 mg·L−1) of the heparin sample was added to 200 μL of a 10 mg·L−1 aqueous Azure A solution. Absorbance was then measured at 645 nm. Sulfation rate and DS were calculated from a calibration curve constructed using values of absorbance obtained from a serial dilution (0 to 30 mg·L−1) of a dextran sulfate standard (Sigma-Aldrich, sulfur content of 17%). 3157

DOI: 10.1021/acs.biomac.7b00797 Biomacromolecules 2017, 18, 3156−3167

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Biomacromolecules Microwave-Assisted Synthesis of HEP-IONP. A 1 mL solution of iron-(III) chloride hexahydrate FeCl3·6H2O (17.5 mg, 0.7 mmol) was mixed with UFH, enoxaparin sodium, or usHEP (100 mg) dissolved in distilled water (1.25 mL) in a microwave-adapted flask. Immediately after, hydrazine monohydrate (N2H4, 64−65%) was added and the tube was rapidly sealed before being placed in the microwave unit (CEM Discover SP). The solution was ramped to 100 °C in 60 s with the irradiating power set at 300 W, and magnetic stirring was allowed at this temperature for a further 3 min 45 s. Subsequently, the sample was cooled down at RT using the instrument air cooling system. HEP-IONP solution was purified twice by gel filtration in a PD10 desalting column (GE Healthcare) to remove hydrazine, unreacted organic iron precursors, and a part of the unabsorbed heparin surfactant. The remaining side products were next discarded by ultrafiltration (3500 rpm, 10 min) with an Amicon Ultra Centrifugal Filter (cutoff: 50 kDa, 2 mL, Merck Millipore), and pure HEP-IONP were redispersed in Milli-Q water for further physicochemical characterizations and bioactivity assays. Physicochemical Characteristics of HEP-IONP. HEP-IONP hydrodynamic size and polydispersity index (PIHEP‑IONP) were measured with a Nano S90 (Malvern Instruments, U.K.) using 12 mm2 polystyrene cuvettes, and the zeta potential was measured with a Nano ZS90 (Malvern Instrument, U.K.) using folded capillary cells. Morphology and core size were determined using a 200 kV JEOL JEM 2011 transmission electron microscope (Jeol Ltd. Japan) equipped with an Oxford-INCA EDS (energy dispersive spectroscopy) system. Crystallographic analyses were performed by selected area electron diffraction (SAED). Dark-field imaging (in a parallel beam mode) was used to highlight the nanometer-sized crystalline particles. The objective aperture was placed in the diffraction plane allowing only low-index diffracted beams from iron oxide phases to form the image. For sample preparation, one drop of a diluted magnetic nanoparticle suspension was placed on a carbon-coated copper grid (Holey, Oxford Instrument) and water was allowed to evaporate at RT for 24 h. Mean size and standard deviation were calculated by counting about 150 particles. Fourier transform infrared spectroscopy (FTIR) spectra were obtained on a Perkin-Elmer Spectrum 400 Series spectrometer (Perkin−-Elmer, USA) with 0.2 mg of lyophilized samples; each spectrum was obtained by averaging 32 interferograms with a resolution of 1 cm−1. Thermogravimetric analysis (TGA) spectra were obtained with a Setsys Evolution (Setaram, France). The lyophilized HEP-IONP and free heparins were heated from 20 to 1000 °C at a rate of 10 °C·min−1 under argon gas at a flow of 20 mL·min−1. Measurements of Heparin Content in the Coatings of HEP-IONP. The heparin content in HEP-IONP was determined by measuring the sulfation rate with the colorimetric “Azure A” method as previously described. Absorbance read at 645 nm of serial dilutions of each HEP-IONP was compared to a calibration curve constructed with a serial dilution (0 to 30 mg·L−1) of the free heparin corresponding to that coating of the IONP. Absorbance spectra of the three HEP-IONP solutions were performed as controls to confirm there was no interference between the Azure A coloration and the IONP absorbance read at 645 nm. The concentrations of heparin coating the IONP thus determined were compared to the results of the TGA analysis. Cell Viability Assays. The impact of HEP-IONP and HEP on the viability of human embryonic kidney cell cultures (HEK 293, obtained from ATCC) was assessed using Alamar Blue assay. Cells were seeded into a 96-well plate at a density of 10,000 cells/well in 50 μL of complete OptiMEM medium (10% FBS, 1% penicillin/streptomycin) and incubated for 24 h at 37 °C. On day 2, 40 μL of complete OptiMEM medium containing different concentrations of HEP or HEP-IONP (20, 50, and 100 μg·mL−1 of heparin products either free or in the IONP coating) was added into the wells. Concentrations were tested in eight replicates. The plates were then incubated for a further 24 or 48 h at 37 °C. Finally, 10 μL of commercial Alamar Blue solution (Life technology) was added into each well and the plate was incubated for a further 3 h at 37 °C. Each well supernatant was then transferred into a black polystyrene plate (Corning #3915) and fluorescence recorded on a BMG Labtech Fluostar Omega spectrofluorometer at λem = 590 nm after excitation at λem = 544 nm. Cell viability was calculated as a percentage of fluorescence obtained normalized to one of the unexposed

control cells (treated with vehicle). The positive control for cytotoxicity consisted of a 20% DMSO aqueous solution. For visualization of HEP-IONP uptake by HEK 293, cells were first seeded into a 24-well plate at a density of 2000 cells/well and allowed to grow for 3 days in a complete OptiMEM medium. Next, fresh medium containing HEP-IONP (at a concentration of 200 μg·mL−1 of heparin products present in the IONP coating) was added into the wells and incubation was followed for a further 6 h. At the end, cells were washed with PBS (pH 7.4) before fixation with paraformaldehyde (4%) for 10 min. The fixed cells were then stained with a mixture of potassium ferrocyanide solution and hydrochloric acid solution (2%) (1:1) for 10 min. After PBS washing, the cells were finally counterstained with a nuclear fast red solution for 5 min before microscopy. All reagents used came from an iron stain kit purchased from Abcam (ab150674). Magnetic Characterization of HEP-IONP. Sample magnetic measurement was performed in a vibrating sample magnetometer using 100 μL of HEP-IONP solution in a special sample holder. Magnetization curves were recorded at RT first by saturating the sample in a field of 1 T. The magnetization values were normalized to the amount of iron to yield the specific magnetization (emu·g−1 of Fe). The initial susceptibility (χ) of suspensions was measured in the field range ±100 Oe, and the saturation magnetization values (Ms) were evaluated by extrapolating to infinite field the experimental results obtained in the high field range in which the magnetization linearly increased with 1/H. For determination of the NMR relaxometric values, the T2 and T1 relaxation times of a series of HEP-IONP dilutions were measured in a Bruker MQ60 (Bruker Biospin, Germany) at 1.5 T and 37 °C. Acquisition parameters were fixed to suit most of the samples for a similar dynamical range of the spectrometer. Bruker’s curve-fitting monoexponential tool was used to find the corresponding T2 values using a Carr−Purcell−Meiboom−Gill (CPMG) pulse sequence. Interpulse tau was changed between 0.05 and 5 ms, TR = 5 s, and 100−200 echoes were used. Similarly, T1 values were obtained using an inversion recovery (IR) sequence and the monoexponential curve-fitting option. Inversion times were adjusted accordingly: first, TI = 5 ms, last TI = 20,000 ms, TR = 15 s, 4 averages, 10 points. The longitudinal relaxation rate R1 (1/T1, s−1) and transversal relaxation rate R2 (1/T2, s−1) were obtained from the measured relaxation times. Linear fitting of these data related to the iron concentration (mM) provided straight lines whose slopes corresponded to relaxivities (r1 and r2 in s−1·mM−1) according to the equation Ri = Rbi + ri [Fe] (i = 1, 2). The iron concentration of the HEP-IONP was calculated using a NMR relaxometry method on the basis of the relaxivity of Fe3+ iron ions in solution.51 Briefly, HEP-IONP solutions (100 μL) were first dissolved in a H2O2/HNO3 mixture overnight at 30 °C. The iron concentrations of the solutions were obtained measuring the relaxation time T1 and 3+ comparing it with the relaxivity of Fe3+ iron ions (r1Fe ) calculated from a standard curve. This last was constructed plotting the relaxation rate (R1 = 1/T1) as a function of Fe3+ concentrations (0−2 mM) obtained from dilutions of FeCl3·6H2O prepared under the same acidic con3+ ditions as the samples. The relaxivity r1Fe was then deduced from the slope of the linear fitting of data. In Vivo Magnetic Resonance Angiography with HEP-IONP. Mice were housed in specific facilities (pathogen-free for mice) at the Centro Nacional de Investigaciones Cardiovasculares. Experimental procedures were approved by the local Animal Care and Ethics Committee and regional authorities. In vivo MRI in mice was performed with an Agilent/Varian scanner (Agilent, Santa Clara, USA) equipped with a DD2 console and an active-shielded 205/120 gradient insert coil with 130 mT·m−1 maximum gradient strength and a combination of a volume coil and a two-channel phased-array (Rapid Biomedical GmbH, Rimpar, Germany). Female C57BL/6 mice (8 weeks old) were anesthetized with 2% isoflurane (Abbott) and oxygen, and positioned on a mouse bed with constant monitoring of respiratory cycle (SA Instruments, NY). Ophthalmic gel was placed in their eyes to prevent retinal drying. Baseline images were acquired before intravenous (IV) administration of the probes. MRI data were acquired at 60, 180, and 210 min after injection. For MRI angiography, 3D gradient echo with a magnetization transfer contrast adiabatic prepulse was performed with 3158

DOI: 10.1021/acs.biomac.7b00797 Biomacromolecules 2017, 18, 3156−3167

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Biomacromolecules the following parameters: min TR, 11 ms; min TE, 1.75 ms; flip angle, 20°; 3 averages; acquisition matrix, 256 × 128 × 128; MTC flip angle, 810°; duration, 6 ms; offset frequency, 2000 Hz. Comparison of the Anticoagulant Activity of the Free Heparins versus the HEP-IONP. For anti-Xa and anti-IIa activity assays, different concentrations of free heparins or HEP-IONP solutions (25 μL in the range 0.005−1 mg·mL−1) were incubated with antithrombin III (25 μL, 0.625 μg·μL−1) at 37 °C in 96-well plates for 2 min. Then, factor Xa or factor IIa was added at a final concentration of 11.25 nKat·mL−1 (25 μL). After 30 s of incubation, 3.25 nM (25 μL) factor Xa chromogenic substrate (CBS 31.39; CH2SO2-D205 Leu-GlyArg-pNA, AcOH) for the anti-Xa activity assay or 1.4 nM (25 μL) factor IIa chromogenic substrate (CBS 61.50; EtM-SPro-Arg-pNA, AcOH) for the anti-IIa activity assay were added. Absorbance of the reaction mixture was read for 3 min at 405 nm every 4 s with an absorbance reader (FLUOstar Omega BMG Labtech).48 The initial velocity was determined as the slope of the linear segment of the kinetics curve, and the % of inhibition was calculated on the basis of the initial velocity of a blank without inhibitor (PBS). The different IC50 values were obtained from dose−response curves by a classical nonregression analysis (Hill, four parameters). Anti-Xa and IIa activities expressed in units per mL were obtained from a UFH standard curve ranging from 0.1 to 2 U·mL−1. The anticoagulant activities of the different HEP-IONP and corresponding heparins alone were determined by measuring the activated partial thrombloplastin time (APTT), prothrombin time (PT), and thrombin time (TT). All of the assays were carried out using a START 4 coagulometer and assay kits from Stago (France) according to the manufacturer’s instructions. Briefly, 90 μL of normal plasma (Stago, France) was mixed with 10 μL of 0.9% NaCl dilution of the tested sample. For TT assays, the mixture was incubated for 1 min at 37 °C and 100 μL of TT triggering assay reagent was added before recording TT. For APTT assays, 100 μL of APTT assay reagent was added to the mixture prior to incubation for 3 min at 37 °C. Then, 100 μL of 0.025 M CaCl2 triggering solution was added before recording APTT. For PT assays, the mixture was incubated for 2 min at 37 °C and 200 μL of PT triggering assay reagent was added before recording PT. The standard clotting times in all assays were measured using a 0.9% NaCl control solution. Comparison of the Anti-Heparanase Activity of the Free Heparins versus the Heparin Coated Iron Oxide Nanoparticles (HEP-IONP). Inhibition of heparanase activity was assessed using the heparanase assay toolbox (Cisbio Assay, France) and heparanase purchased from R&D systems (HPSE-1 human recombinant heparanase). Briefly, a biotin-HS-Eu-(K) substrate (heparan sulfate labeled with both biotin and Eu3+ cryptate) produces a fluorescent emission at 665 nm by fluorescence resonance energy transfer (FRET) upon addition of streptavidin-XL665 (SA-XL665). Active heparanase cleaves the substrate, resulting in a loss of energy transfer, and thus a reduction in SA-XL665 emissions. Enzymatic reaction was performed in white 96-well half area plates (Corning #3693) and monitored using a spectrofluorometer (BMG Labtech FLUOstar Omega) with a high timeresolved fluorescence (HTRF) module. The reaction was assayed with or without heparin products at 37 °C in a final volume of 60 μL. First, 15 μL of inhibitor solutions in Milli-Q water was added into the wells followed by 15 μL of heparanase solution (HPSE-1, 400 ng·mL−1 in Tris−HCl at pH 7.5, 0.15 M NaCl and 0.1% CHAPS). After a 10 min preincubation at 37 °C, an enzyme reaction was initiated by adding 30 μL of Biotin-HSEu-(K) (1.0 ng·μL−1 in 0.2 M sodium acetate buffer, pH 4.5) and the plate was incubated at 37 °C for 15 min. The reaction was activated by adding 30 μL of streptavidin-XL665 containing an UFH solution (SA-XL665, 10 ng·μL−1 in NaPO4 0.1 M buffer, pH 7.4, 0.8 M KF, 0.1% BSA and 2 mg·mL−1 UFH). The fluorescence was measured after 2 min at λem1 = 620 nm and λem2 = 665 nm, after 60 μs of excitation at λex = 337 nm. The Delta F (%) was calculated using the following equation as indicated by the suppliers, and the % of inhibition was calculated on the basis of the Delta F (%) of heparanase activity without inhibitor

where F665 and F620 were fluorescence signals measured at 665 and 620 nm, respectively. Final IC50 values were determined on the basis of the curve fitting tool from SigmaPlot software (Systat Software Inc., San Jose, USA) using a sigmoidal, logistic, three-parameter equation. To monitor the heparin compound depolymerization due to the heparanase activity, UFH and UFH-IONP were incubated with heparanase (2.2 μg·mL−1) at 37 °C in a final volume of reaction buffer of 100 μL (0.2 M sodium acetate buffer, pH 4.5, 0.15 M NaCl, and 0.1% CHAPS). Aliquots of 10 μL were collected over time before recording the APTT and TT as described above. Blanks consisted of the enzyme alone in the reaction buffer solution. Concentrations of heparin were, respectively, 0.075 and 0.025 mg·mL−1 for the APTT and TT assays.

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RESULTS AND DISCUSSION Microwave-Assisted Synthesis of Heparin-Coated Iron Oxide Nanoparticles (HEP-IONP). For comparative purposes, three differently heparin coated iron oxide nanoparticles (HEPIONP) were synthesized. We selected native heparin (UFH) and a commercial LMWH (Enoxaparin-Lovenox, LOV) because they are the most commonly reported backbones or coatings for heparin-based nanocarriers, and also the references in terms of anticoagulant activity.14 A LMWH prepared by ultrasoundassisted radical hydrolysis depolymerization and previously presented by our group as a potential antitumor candidate (usHEP) was also assessed.48 The main physicochemical characteristics of the three heparins are presented in Table 1. Table 1. Main Physicochemical Features of the Heparins Used for the Preparation of IONPa UFH LOV usHEP

Mn (Da)

Mw (Da)

DP

PIHEP

DS

11,130 4299 ± 120 2479 ± 181

12,237 5535 ± 182 5759 ± 230

46 18 10

1.09 1.28 2.32

1.26 1.2 0.68

a

UFH, unfractionated heparin; LOV, Enoxaparin-Lovenox; usHEP, LMWH obtained after 5 h of ultrasound-assisted radical hydrolysis depolymerization; Mn (Da), average molecular weight; Mw (Da), weight average molecular weight; DP, degree of polymerization; PIHEP, polydispersity index of heparins; DS, degree of sulfation.

HEP-IONP were prepared by adaptation of an ultrarapid microwave (MW)-assisted method reported for the synthesis of dextran-coated IONP.38 Briefly, the general procedure involved a one-step hydrazine-mediated reduction of iron-(III) chloride hexahydrate salt in the presence of heparins used as a surface stabilizing coating. The mixture was heated for 3 min 45 s using a very rapid ramping (60 s) to 100 °C by MW irradiation at a power of 300 W. The solution was purified by gel filtration to discard all of the unreacted iron precursor plus hydrazine, and the excess of heparin was then removed by ultrafiltration (cutoff: 50 kDa) to obtain a pure stable colloidal suspension of HEP-IONP in water. The different variables in the reaction were optimized according to the previously published literature46,53−56 and by subsequent empirical adjustments to obtain good quality iron oxide cores, while maintaining optimum colloidal stability. The surfactant was added in large excess, above a required minimum stoichiometric ratio to iron, below which the nanoparticles were observed to not be stable or much larger. The sample quality was also particularly sensitive to the microwave reaction time, and minimal changes (±10 s) led to unsuccessful attempts or important increases in hydrodynamic size. The objective here was to obtain HEP-IONP as small as possible, keeping in mind this will improve the magnetic properties for T1-MRI contrast and in vivo behavior. Larger hydrodynamic

Delta F (%) = {[(F665/F620)sample − (F665/F620)blank ]/(F665/F620)blank } × 100 3159

DOI: 10.1021/acs.biomac.7b00797 Biomacromolecules 2017, 18, 3156−3167

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Biomacromolecules

Figure 1. Main physicochemical features of HEP-IONP. (A) TEM pictures with SAED patterns in the inset, (B) hydrodynamic sizes, (C) TGA analysis, and (D) FTIR spectra.

Table 2. Main Physicochemical Features of HEP-IONP UFH-IONP LOV-IONP usHEP-IONP

Z-average (nm)

PIHEP‑IONP

zeta potential ζ (mV)

weight ratio (HEP/Fe)

r1 and r2 relaxivities (mM−1·s−1); (r2/r1)

55.7 43.5 29.5

0.15 0.16 0.23

−40 −51 −49

15.7:1 2.3:1 2.6:1

r1 13.3; r2 73.1; (5.5) r1 9.5; r2 58.8; (6.1) r1 4.0; r2 12.8; (3.3)

than −40 mV were obtained and indicated the presence of negative charges exposed on the surface of coated IONP (Table 2). This ensured an excellent colloidal stability for the three HEP-IONP tested due to strong repulsive electrostatic interactions. TGA was performed on each HEP-IONP and its corresponding free heparin to evaluate the graft density of polymeric coating. As shown in Figure 1C, the weight loss of HEP-IONP up to 750 °C corresponded to the main degradation of the organic coating. As IONP are stable below this temperature, after taking into account the remaining weight contribution of heparins from their analysis, we found that the organic heparin coating represented a large part of IONP total weight, ranging around 94, 73, and 70% for UFH-IONP, LOV-IONP, and usHEP-IONP. This indicates a thick coating layer and explains the difference between the relatively large hydrodynamic sizes of the probes compared to their iron oxide core sizes. Estimated heparin/iron oxide weight ratios for HEP-IONP were consistent with the Mw of each type of heparin belonging to their coating, with larger polymers contributing more to the nanostructure total weight (Tables 1 and 2). Rough gravimetric data were then refined with heparin content measurements by Azure A assay (Figure S4). The obtained data ranges supported the previous gravimetric estimation and were further used as a concentration reference of heparin content in the IONP coatings for the biological tests. Then, we characterized the structural composition of surface coatings. Heparin structures should be reasonably preserved

sizes obtained with different conditions may indicate that a higher amount of coated heparins can be regulated (data not shown) but at the expense of this purpose.29 Further experiments shall be conducted to determine the exact influence of the different parameters on the hydrodynamic and core sizes as well as the amount of coated heparins. Physicochemical Characterization of the HeparinCoated Iron Oxide Nanoparticles (HEP-IONP). Transmission electron microscopy (TEM) in bright and dark-field imaging revealed small, well-dispersed iron oxide cores (Figure 1A and Figure S1) of around 5.4 ± 1.8 nm diameter for UFH-IONP, 5.4 ± 1.5 nm for LOV-IONP, and 4.6 ± 0.9 nm for usHEP-IONP (N = 150, Figure S2). SAED patterns confirmed the good crystallinity of the cores (insets of Figure 1A and Figure S3). As determined by dynamic light scattering (DLS), the hydrodynamic size (Z-average) in water of UFH-IONP, LOV-IONP, and usHEP-IONP was, respectively, 55.7, 43.5, and 29.5 nm (Figure 1B) and remained stable for weeks (data not shown). HEP-IONP polydispersity indexes (PIHEP‑IONP) were all below 0.25, the standard to consider a homogeneous dispersion. Results showed the expected increase in hydrodynamic size scaling with the length of the type of heparin used. To assess colloidal stability, the zeta potential ζ was measured at physiological pH. Heparin polymers are negatively charged due to the multiple carboxylic and sulfate groups present on their disaccharide units. Accordingly, high negative potentials all less 3160

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Article

Biomacromolecules

Figure 2. Main magnetic properties of HEP-IONP. (A) Magnetization curves at 298 K (1 Oe = 1000 T). (B) Plot of the transversal (T2) and longitudinal (T1) relaxation rates vs iron concentration of HEP-IONP.

Magnetic Properties of Heparin-Coated Iron Oxide Nanoparticles (HEP-IONP) as MRI Contrast Agent. HEPIONP magnetic properties were first analyzed using a vibrating sample magnetometer (VSM). They all displayed a superparamagnetic behavior (no hysteresis loop) with a low saturation magnetization value (MS) between 30 and 40 emu·g−1 (Figure 2A). This small value was anticipated given the small size and the lack of alignment of spins/magnetic moments on the nanoparticle surface. To assess MRI performances of HEP-IONP used as potential contrast agents, longitudinal (r1) and transversal (r2) relaxivities were investigated at 37 °C and 1.5 T, the field strength of most clinical scanners. Optimum parameters for T1-MRI contrast agents consist of a high r1 with the lowest possible r2/r1 ratio to induce the least amount of T2* effect relative to the T1 shortening effect. Standards are the paramagnetic gadolinium (Gd) chelate-based agents such as Dotarem or Magnevist with an r1 value of about 3 mM−1·s−1 and r2/r1 ratio near 1. However, these agents present risks of adverse effects, including nephrotoxicity or Gd deposition in organs. As an alternative, IONP can also produce T1 contrast, which comes from the unique state of iron atoms in the NP surface layer that leads to disordered magnetic spins, i.e., the spin canting effect.41 Usual IONP of large hydrodynamic size >50 nm (like Feridex or Resovist, ferrofluids made of polydispersed core aggregates) or large iron oxide cores >8 nm are nonetheless only used as T2 agents because of a markedly high transversal relaxivity value due to their strong magnetization, resulting in an unfavorable r2/r1 ratio.45 However, the latter can be adjusted by adapting the size, composition, and surface state of the NP. For instance, smaller hydrodynamic sizes will decrease the r2 value by dropping the magnetization provoked by the aggregation of the cores. Thus, various IONP with hydrodynamic size