Family of Bioactive Heparin-Coated Iron Oxide Nanoparticles with

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Family of bioactive heparins-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, Frederic Sannier, Stéphanie Bordenave, Jean-Marie Piot, Jesús Ruiz-Cabello, Ingrid Fruitier-Arnaudin, and Thierry Maugard Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00797 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Family of bioactive heparins-coated iron oxide nanoparticles with positive contrast in magnetic resonance imaging for specific biomedical applications Hugo Groult1, Nicolas Poupard1, Fernando Herranz2, Egle Conforto3, Nicolas Bridiau1, Frederic Sannier1, Stéphanie Bordenave1, Jean-Marie Piot1, Jesús Ruiz-Cabello2, Ingrid Fruitier-Arnaudin1 and Thierry Maugard1,*. 1 UMR CNRS 7266 LIENSs, Approches Moléculaires Environnement-Santé environnement (AMES), University of La Rochelle, France. 2 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain. 3 UMR CNRS 7356 LaSIE - University of La Rochelle, France. KEYWORDS. Heparins - Iron oxide nanoparticles – T1-weighted magnetic resonance imaging – Heparanase – Anticoagulant – Anti-tumor.

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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 proof of concept, magnetic resonance angiography (MRA) were succeeded in mice up to 3 hours after intravenous administration. This new IONP-based positive contrast appropriate for clinic together with the long vascular circulating times can enabled 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 anti-heparanase 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 anti-tumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.


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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 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 are 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, an 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


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molecules and facilitates tumor cells extravasation.11 Although the application of heparins as anticancer agents has long 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 anti-angiogenic and anti-metastatic 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-glycine-aspartic 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, bio-acceptability 25,26 and hemocompatibility.27–29 They have also been described as a suitable coating to load bioactive agents on IONP surface, such as chemotherapeutics 30,31, proteins 32,33, virus 34 or stem cells 35–37


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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 these approaches, iron oxide cores have been synthesized with the traditional co-precipitation method and UFH or LMWH have then been adsorbed on the surface 25,33 otherwise attached to a pre-existing coating either covalently 28,32 or electrostatically.27 In addition to the cumbersome multiple-step 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 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 agent 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 non-toxicity, 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 to reduce considerably the reaction times, increase yields and enhance reproducibility over other approaches.46 IONP with dextran coating produced by MW technology have lately been proposed as suitable multi-functional T1-MRI contrast agents.38,47


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Here, a one-step microwave-assisted method for the rapid synthesis of IONP was adapted to produce heparins 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. 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 an hydrogen peroxide catalyzed radical hydrolysis assisted by ultrasounic waves, as previously described. 48 Briefly, UFH (100 mg) was first resuspended in 4 mL of milliQ 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 0.5s with a frequency of 30 kHz in a Radleys® reactor for 5 hours at room temperature (RT). Solution was finally dialyzed (cut-off: 1kDa) and lyophilized to obtain pure usHEP (80 mg, 80% yield).


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Structural and quantitative analysis by size exclusion chromatography. Structural and quantitative analyzes 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 TSKGEL G3000PW and two TSK-GEL G2000PW columns mounted in series. The columns were maintained at 30°C and the products were eluted with 0.1M Sodium nitrate (NaNO3) at a flow rate of 0.5 mL.min-1. The products were detected and quantified by differential refractometry using HP Chemstation software off-line for the processing. Heparin oligosaccharides of different molecular weights purchased from Iduron (Manchester, UK) 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 equations.49 Mn = (Σ Ni × Mi) / Σ Ni; Mw = (Σ Ni × Mi2) / (Σ Ni × Mi); DP = Mn / M0 ; PI = Mw / Mn 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 degree of sulfation of heparins. 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 were added to 200 µL of a 10 mg.L1

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%).


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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 irradiating power set at 300 W and allowed magnetic stirring at this temperature for 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 Amicon Ultra Centrifugal Filter (cut-off: 50 kDa, 2 mL, Merck Millipore) and pure HEP-IONP were redispersed in milliQ 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, UK) using 12-mm2 polystyrene cuvettes and zeta potential was measured with a Nano ZS90 (Malvern Instrument, UK) 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


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nanoparticle suspension was placed on a carbon-coated copper grid (Holey®, Oxford Instrument) and water was allowed to evaporate at RT for 24h. 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°C to 1,000°C at a rate of 10°C.min-1 under argon gas at a flow of 20 mL.min-1. Measurements of heparins 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 HEPIONP 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 HEPIONP solutions were performed as controls to confirm there was no interference between the Azure A coloration and the IONP absorbance read at 645nm. The concentrations of heparin coating the IONP thus determined were compared to the results of the TGA analysis. Cell viability assays. 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 24h 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) were


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added into the wells. Concentrations were tested in 8 replicates. The plates were then incubated for a further 24h or 48h 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 3h at 37°C. Each well supernatant was then transferred into a black polystyrene plate (Corning® #3915) and fluorescent 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 the one of the unexposed control cells (treated with vehicle). The positive control for cytotoxicity consisted in 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 cell/well and allowed to growth for 3 days in a complete OptiMEM® medium. Next, fresh medium containing HEP-IONP (at a concentration of 200 µg.ml-1of heparin products present in the IONP coating) were added into the wells and incubation was followed for further 6 hours. At the end, cells were washed with PBS (pH = 7.4) before fixation with paraformaldehyde (4%) for 10 minutes. 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 Characterisation 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 Fe). The initial susceptibility (χ) of suspensions was measured in the field range ± 100 Oe, and the saturation magnetization values (Ms) were evaluated by


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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 mono-exponential tool was used to find the corresponding T2 values using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. Inter pulse tau was changed between 0.05 to 5 ms, TR = 5s, and 100-200 echoes were used. Similarly, T1 values were obtained using an Inversion Recovery (IR) sequence and the mono-exponential curve- fitting option. Inversion times were adjusted accordingly: first TI = 5ms, last TI = 20,000ms, TR = 15s, 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 the equation Ri=Rbi+ ri [Fe], (i= 1,2). Iron concentration of the HEP-IONP was calculated using a NMR relaxometry method based on 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 concentration of the solutions were obtained measuring the relaxation time T1 and comparing it with the relaxivity of Fe3+ iron ions (r1Fe3+) 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 in the same acidic conditions as the samples. The relaxivity r1Fe3+ was then deduced from the slope of the linear fitting of data.


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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 2-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 (i.v.) administration of the probes. MRI data were acquired at 60, 180 and 210 minutes after injection. For MRI angiography, 3D gradient echo with a magnetization transfer contrast adiabatic pre-pulse was performed with the following parameters: min TR, 11 ms; min TE, 1.75 ms; flip angle 20; 3 averages; acquisition matrix 256 × 128 x 128; MTC flip angle 810 deg; 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 of 0.005 to 1 mg.ml-1) were incubated with anti-thrombin III (25 µL, 0.625 µg.µL-1) at 37°C in 96-well plates for 2 minutes. 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-Gly-Arg-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


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reaction mixture was read for 3 min at 405 nm every 4 s with an absorbance reader (FLUOstar Omega BMG Labtech) 52. The initial velocity was determined as the slope of the linear segment of the kinetics curve and the % of inhibition was calculated based on the initial velocity of a blank without inhibitor (PBS). The different IC50 were obtained from dose-response curves by a classical non-regression analysis (Hill, 4 parameters). Anti-Xa and IIa activities expressed in unit 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 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) were 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 were added before recording TT. For APTT assays, 100 µL of APTT assay reagent were added to the mixture prior incubation for 3 min at 37°C. Then, 100 µL of 0.025 M CaCl2 triggering solution were 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 were 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


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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 time resolved 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 milliQ water were added into the wells followed by 15 µL of heparanase solution (HPSE-1, 400 ng.ml-1 in Tris-HCl at pH 7.5, 0.15M NaCl and 0.1% CHAPS). After a 10-min pre-incubation at 37°C, an enzyme reaction was initiated by adding 30 µL of Biotin-HSEu(K) (1.0 ng.µL-1 in 0.2M 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 0.1M buffer, pH 7.4, 0.8M KF, 0.1% BSA and 2 mg.mL-1 UFH). The fluorescence was measured after 2 minutes at λem1=620 nm and λem2=665nm, 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 based on the Delta F(%) of heparanase activity without inhibitor: Delta F (%) = {[(F665/F620)sample-(F665/F620)blank] / (F665/F620)blank }x 100 Where: F665 and F620 were fluorescence signals measured at 665 nm and 620 nm, respectively. Final IC50 were determined based on the curve fitting tool from SigmaPlot software (Systat Software Inc, San Jose, USA) using a sigmoidal, logistic, 3-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


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buffer of 100 µL (0.2M sodium acetate buffer, pH 4.5, 0.15M NaCl and 0.1% CHAPS). Aliquots of 10 µL were collected over time before recording the APTT and TT as described above. Blanks consisted in the enzyme alone in the reaction buffer solution. Concentrations of heparin were respectively of 0.075 and 0.025 mg.ml-1 for the APTT and TT assays. RESULTS AND DISCUSSION Microwave-assisted synthesis of heparins coated iron oxide nanoparticles (HEP-IONP). For comparative purposes, three differently heparin coated iron oxide nanoparticles (HEP-IONP) were synthesized. We selected native heparin (UFH) and a commercial LMWH (EnoxaparinLovenox ®, 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 ultrasound-assisted radical hydrolysis depolymerization and previously presented by our group as a potential anti-tumor 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 IONP. Mn (Da)

Mw (Da)

DP

PIHEP

DS

UFH

11,130

12,237

46

1.09

1.26

LOV

4,299 ± 120

5,535 ± 182

18

1.28

1.2

usHEP

2,479 ± 181

5,759 ± 230

10

2.32

0.68

UFH, unfractionated heparin; LOV, Enoxaparin-Lovenox®; usHEP, LMWH obtained after 5h 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 ultra-rapid microwave (MW)-assisted method reported for the synthesis of dextran-coated IONP.38 Briefly, the general procedure involved a


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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 the unreacted iron precursor plus hydrazine, and the excess of heparin was then removed by ultrafiltration (cut-off: 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 literature 46,53–56 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 not stable or much larger. The sample quality was also particularly sensitive to the microwave reaction time, and minimal changes (± 10 seconds) 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 sizes obtained with different conditions may indicate that 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 amount of coated heparins. Physicochemical characterization of the heparins coated iron oxide nanoparticles (HEPIONP).


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Figure 1. Main physicochemical features of HEP-IONP. A) TEM pictures with SAED patterns in inset, B) Hydrodynamic sizes, C) TGA analysis and D) FTIR spectra. 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 of 55.7, 43.5 and 29.5 nm (Figure 1B) and remained stable for weeks (data not shown). HEP-IONP polydispersity indexes


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(PIHEP-IONP) were all below 0.25, the standard to consider an 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 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 were 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 explain 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, larger polymers contributing more to the nanostructure total weight (Table 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 heparins content in the IONP coatings for the biological tests. Then, we characterized the structural composition of surface coatings. Heparin structures should be reasonably preserved during MW-assisted synthesis


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although hydrazine treatment has previously been reported to induce N-deacetylation of various polysaccharides.57,58 For each UFH-IONP, LOV-IONP and usHEP-IONP, the presence of their respective heparin was then checked by FTIR. All HEP-IONP spectra showed the representative bands of heparin backbone at 3,000-3,650 cm-1 (O-H), 2,900 cm-1 (C-H), 1,600 cm-1 (νs C=O), 1,400 cm-1 (N-H) and 1,200/1,000 cm-1(S=O) together with the typical signals of iron oxide for instance at 400 cm-1 (Figure 1D).28 In addition, the N-C band corresponding to N-acetyl was detected in all cases, while no vibration of unsubstituted N-H (two stretch bands in the 3,1003,500 cm-1 range) was seen. This is a first confirmation that the MW-assisted method for IONP synthesis did not meet the conditions for hydrazinolysis. Finally, the potential cytotoxicity of HEP-IONP was evaluated in human embryonic kidney cells 293 (HEK 293). Cell viability was determined after incubation of each HEP-IONP with HEK 293 cells over 48 hours at three different heparin doses (20, 50 and 100 µg.ml-1) that correspond to iron doses in the 0-50 µg.ml-1 range according to the HEP/iron ratio calculated by TGA of each HEP-IONP. For comparison cell viability assay was also performed with the corresponding free heparins at the same doses. After 48 h, no toxic effects more than the ones already reported for UFH and LOV at high doses were observed.59 A slightly more pronounced effect was noticed at the higher dose for the HEP-IONP after 24 h incubation but that was resorbed at 48 h (Figure S5). These preliminary data suggested that the HEP-IONP can be safely used in vivo at the therapeutic heparin levels currently employed. This was expected as UFH and LMWH are already commercial or currently in clinical phases and several IONP have been as well approved by FDA.60 Magnetic properties of heparins coated iron oxide nanoparticles (HEP-IONP) as MRI contrast agent. HEP-IONP magnetic properties were first analyzed using a vibrating sample


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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 in 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) chelates-based agents such as Dotarem ® or Magnevist ® with an r1 value 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 alternative, IONP can as well produce T1 contrast, which come 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 cores 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 r2 value by dropping the magnetization provoked by the aggregation of the cores. Thus, various IONP with hydrodynamic size < 50 nm have been used as both T2 and T1 contrast agents. An example is the commercial IONP-based agent Ferumoxytol® (r1 of 15 mM-1.s-1, r2 of 89 mM-1.s-1, r2/r1 of 5.9 at 1.5 T), an iron replacement product for anemia, that show also great promise in clinical studies for diagnosis based on positive contrast T1-weigthed MRI.61,62 Most effective to enhance the T1


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contrast effect is the reduction of the core size. While r1 is slightly affected, r2 value is strongly reduced as the magnetic moment of IONP rapidly decrease due to the reduction of the volume of the magnetic anisotropy and spin disorders at the surface.39 Therefore, it appears in the state-ofart very small and exceedingly small IONP (VS-IONP/ES-IONP) with cores < 5 nm and r2/r1 in the 2.0-4.5 range, as first-rates IONP-based T1contrast agents.41 Even, nearly paramagnetic properties have been achieved with size below 1.8 nm39,63 or using different iron oxide NP phase showing antiferromagnetic properties.64,65 Measurements for HEP-IONP are presented in Figure 2B and Table 2. As expected, usHEP-IONP displayed a little lower r1 of 4.0 mM-1.s-1 but much lower r2 of 12.8 mM-1.s-1 than UFH-IONP and LOV-IONP (r1 in the 9.5-13.5 mM-1.s-1 range, r2 in the 55-75 mM-1.s-1 range) because of the smaller core as well less polydispersed, 4.6 ± 0.9 nm compare to 5.4 ± 1.8 nm. This makes usHEP-IONP as the most ideal candidate for T1 imaging with the smallest r2/r1 ratio of 3.2. For the two other probes, a template effect from the heparin coating of higher molecular weight may occur, as corroborated by the increase of their hydrodynamic size and previously reported by Ternent et al. with native heparin.29 This somehow aggregation is known to increase the transverse relaxivity then explaining the consequent higher r2/r1 ratio obtained for UFH-IONP and LOV-IONP (respectively 5.5 and 6.1), and has been already described for VS-IONP coated with polyethylene glycol of different length.66 Overall, unsurprisingly the HEP-IONP display a r2/r1 ratio more suitable for T1weighted MRI than the IONP with heparins coatings already proposed in the literature that are only used as T2 contrast agents (see introduction), primarily because of their much larger hydrodynamic and core sizes than the ones succeeded here. Among the class of IONP-based T1 agents, these results stand into those of VS-IONP for usHEP-IONP and into those of commercial Ferumoxytol® for UFH and LOV-IONP, but yet below the ES-IONP performances.40,41


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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 iron concentration of HEP-IONP. Table 2. Main physicochemical features of HEP-IONP. Zaverage

PIHEPIONP

(nm) UFH-IONP

zeta potential ζ (mV)

Weight ratio (HEP/Fe)

r1 and r2 relaxivities (mM-1.s-1) ; (r2/r1)

55.7

0.15

-40

15.7 : 1

r1 13.3; r2 73.1 ; (5.5)

LOV-IONP 43.5

0.16

-51

2.3 : 1

r1 9.5; r2 58.8 ; (6.1)

usHEPIONP

0.23

-49

2.6 : 1

r1 4.0; r2 12.8 ; (3.3)

29.5


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In vivo magnetic resonance angiography with heparins coated iron oxide nanoparticles (HEP-IONP). The value of HEP-IONP for in vivo positive contrast MRI was investigated in mice. After injection in the tail vein of LOV-IONP or usHEP-IONP (2 mg Fe per kg), main vascular architecture was brightened on the magnetic resonance angiography (MRA) acquisitions demonstrating that the nanoparticles can enhance T1 relaxation in the circulating system. The high signal intensity generated images with excellent anatomical details that clearly depict carotids, aorta, heart chambers, main veins and even some smaller vessels (Figure 3). This is remarkable if we consider the images where taken at high field (7 T). usHEP-IONP provided better contrast than LOV-IONP consistently with the relaxometric parameters calculated (higher r1 and lowest r2/ r1 ratio). The bright signal in the vascular system was maintained for more than 210 min post injection for both probes showing possibility for T1 enhanced blood pool MRI. T1 enhanced blood pool MRI can provide high-resolution images from sequences of long acquisition times (steady-state imaging). It is of great interest in clinical MRI for cardiovascular diseases diagnostic (especially myocardial infraction, atherosclerotic plaque and thrombosis), for oncology to characterize the tumor angiogenesis, and for detection of renal failure.39 This possibility offered by the HEP-IONP is particularly advantageous in comparison of the commercial gadolinium complexes based T1-MRI contrast agents which produce an intense contrast but that vanish rapidly because of their short vascular life.40,42 The long blood life of HEP-IONP was derived from the optimal particle small-size and thick biocompatible polymeric layer that avoid their uptake by reticuloendothelial system.67 Because in this study the different heparin coatings are designed to be at the same time the stabilizing surfactant and a specific bioactive group, to obtain a long vascular life time was crucial in order that probes reach the biological target and/or exert its specific pharmacological activity.


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Figure 3. MRI angiography in mice at 60 min, 180 min and 210 min after i.v. administration of A) LOV-IONP and B) usHEP-IONP (2 mg Fe/kg-1). For instance, when anticoagulant properties will be discussed, it is promising to notice that the first estimation about vascular life time of LOV-IONP (signal seen after 210 min) is in the same range than the reported half-life of Lovenox® (4-5 hours in human ; 4000 U in subcutaneous injection).68 To the best of our knowledge, it is the first time that heparin-coated nanoparticles are proposed as a potential T1-MRI contrast agent. It could be of particular interest for angiography in cardiovascular disease or angiogenesis-based tumor diagnosis. We wanted then to demonstrate


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how the different heparin coating might also act as bioactive group. Indeed conjugation or adsorption of biomolecules onto NP surface could result in significant changes (loss/synergistic effect) in their biological effects, so we investigated the anticoagulant and anti-heparanase activities of the prepared HEP-IONP compared to the free heparins corresponding to their coating. Study of HEP-IONP anticoagulant activity in comparison with their corresponding free heparin. We first assessed HEP-IONP anticoagulant activity on the specific antithrombinmediated factors Xa and IIa inhibition by chromogenic in vitro assays. These key factors activate fibrin formation that polymerizes and creates blood clots with thrombocytes. UFH is considered the gold standard anticoagulant that set the unit for assessing anti-Xa and anti-IIa activities. This is mainly due to a specific highly sulfated pentasaccharide sequence within the heparin chain that binds the antithrombin (AT III) to form a complex, which in turn inhibits the coagulation factors Xa and IIa. Regarding factor IIa, an additional steric hindrance effect caused by the other sugars of the chain is involved.6 Heparin molecular weight reduction and desulfation induced by depolymerization or other chemical methods affect the pentasaccharide sequence and decrease the steric effect of the long chain, thus reducing the anticoagulant activity. For instance, LOV is a commercial LMWH anticoagulant that specifically targets the coagulation factor Xa (215 ± 18 U.mg-1) but only slightly inhibits factor IIa (99 ± 5 U.mg-1) because the selective chemical βelimination depolymerization method only decreases the molecular length while preserving the sulfated pentasaccharide sequence.7 Conversely for usHEP, both anti-Xa and anti-IIa activities are reduced because the ultrasound-assisted radical hydrolysis method for depolymerization alters the sequence by random cleavage and desulfation (Table 3).48


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Figure 4 shows the dose-response curves obtained for UFH-IONP, LOV-IONP and usHEPIONP, compared to those of the free heparins used to prepare IONP. All HEP-IONP showed a significant activity in both assays, in most cases slightly lower but within the same range than their respective free heparins in solution. Moreover, similar results were observed for the ability of the different types of heparin to inhibit factors Xa/IIa in HEP-IONP. Thus, UFH-IONP and LOV-IONP anti-Xa activity was higher (246 ± 14 U.mg-1 and 170 ± 18 U.mg-1, respectively) than that of usHEP-IONP (60 ± 4 U.mg-1) while LOV-IONP and usHEP-IONP anti-IIa activity was lower (74 ± 4 U.mg-1 and 52 ± 2 U.mg-1, respectively) than that of UFH-IONP (161 ± 7 U.mg-1, Table 3). This finding supports the fact that the inhibition is not due to the unspecific adsorption of AT III or factors Xa/IIa at the IONP surface. Besides, the anticoagulant assays performed without AT III or with a control IONP devoid of anticoagulant coating did not show any inhibition. These results demonstrated that, depending on their specific structural-functional activity, heparins functionalized to IONP present free active pentasaccharide sequences for AT III activation and the resulting inhibition of factors Xa/IIa. Data in the literature about the anticoagulant activity of heparins coating onto inorganic nanoparticles are highly variable, pointing out a balance between opposite effects.69,70 On the one hand, a strong steric hindrance from the cores could prevent accessibility to heparin active sites, thus reducing its activity. The use of “spacers” such as PEG or PVA between the NP surface and the covalently bound heparin has shown promising results to overcome this effect. 28 On the other hand, the nanoparticlesupported adsorption of the protein of interest increases the affinity for the heparin coating and could enhance the anticoagulant activity. Beneficial effects have also been described regarding the restriction of heparin freedom in a fix conformation, which promotes binding to biological receptors. In our study, we observed increased or equal anticoagulant activity for UFH-IONP in


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comparison to free UFH. This, probably because the long polysaccharidic chains ease the accessibility of AT III to active sequences with minimized steric hindrance from the iron oxide core (in a sense similarly as the “spacers”), allowing the nanoparticle-supported adsorption as predominant input. Such hypothesis may explain as well the relative decrease in anti-Xa and IIa activities of LOV-IONP and usHEP-IONP in comparison to their corresponding free heparin. The accessibility of AT III being in this case more subjected to steric hindrance because the active sequences in these shorter polysaccharide were closer from the nanoparticle core.

Figure 4. IC50 values for anti-factors Xa/IIa activity. A) Anti-factor Xa activity and B) antifactor IIa activity of HEP-IONP and their corresponding free heparins (AT III = 0.625 µg.µL-1 and factor Xa or IIa = 11.25 nKat.mL-1).


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However, these in vitro anti-Xa/IIa assays were biased because the media only contained the proteins of interest. Thus, the nanoparticle-protein interactions promoting the anticoagulant activity were not subjected to competitive adsorption with other proteins as in the plasma. Therefore, we explored HEP-IONP anticoagulant activities on a larger scale using the TT, APTT and PT assays. These tests are in vitro coagulometric measurements of plasma in which the activities of factors of the common, intrinsic and extrinsic coagulation pathways relevant for clinical investigations are assessed. For the three tests, HEP-IONP showed prolonged plasma clotting times similar to the ones of the free heparin belonging to their coating (Figure S6). All reference samples (pure PBS and IONP devoid of anticoagulant coating) showed normal plasma clotting times. In details, for the TT assay, HEP-IONP activities expressed as the concentration required for doubling the standard plasma clotting time (DTC), were similar or slightly higher than those of their corresponding free heparins (Table 3). The largest differences were found for the APTT assay where 2.05 mg.L-1 and 2.80 mg.L-1 were respectively needed for LOV and usHEP to double the clotting time, while only 1.50 mg.L-1 and 1.30 mg.L-1 were respectively needed for their corresponding HEP-IONP. The APTT assay explores the common and intrinsic coagulation pathways, in which both factors Xa and IIa are involved, and is therefore more likely to highlight the difference in activities reported above. Regarding the PT assay, as expected heparins and HEP-IONP had low activities because they are not supposed to interfere in the pathway explored by this test. Differences observed between usHEP-IONP and LOV-IONP and their corresponding free heparin could be due to the unspecific adsorption of cofactors involved in the extrinsic pathway.


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Table 3. Anticoagulant activities of HEP-IONP compared to their corresponding free heparins.

UFH

UFH-IONP

LOV

LOV-IONP

usHEP

usHEP-IONP

Anti-Xa

Anti-IIa

TT

APTT

PT

(U.mg-1)

(U.mg-1)

assay

assay

assay

(IC50 mg.L-1)

(IC50 mg.L-1)

DTC (mg.L-1)

DTC (mg.L-1)

DTC (mg.L-1)

160 ± 5

160 ± 5

0.47

0.88

21

0.052 ± 0.007

0.052 ± 0.002

246 ± 14

161 ± 7

0.38

0.88

25

0.034 ± 0.009

0.052 ± 0.002

215 ± 18

99 ± 5

0.78

2.05

330

0.037 ± 0.003

0.085 ± 0.004

170 ± 18

74 ± 4

0.57

1.30

50

0.049 ± 0.005

0.11 ± 0.006

80 ± 5

92 ± 9

0.70

2.80

90

0.105 ± 0.007

0.091 ± 0.009

60 ± 4

52 ± 2

0.70

1.50

45

0.140 ± 0.009

0.162 ± 0.007

DTc, concentration required for doubling the standard plasma clotting time, error

Family of Bioactive Heparin-Coated Iron Oxide Nanoparticles with

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