Iron sucrose

T1/T2 Dual functional iron oxide MRI contrast agent with super stability and low hypersensitivity benefited by ultrahigh carboxyl group density

ABSTRACT
Clinical acceptable safety and efficacy are the most important issues for the design and synthesis of iron oxide MRI contrast agent. In order to meet the practical requirements, a kind of low molecular weight PAA-coated Fe3O4 nanoparticles (CS015) with super colloidal stability and low hypersensitivity benefited from ultrahigh carboxyl group density were developed in this study. The constitution and physicochemical properties of the particles were characterized by TEM, XRD, FTIR and TGA. Ultrahigh density of COOH on the particles (33 COOH/nm2) was verified while core size (5.1 nm) and dynamic diameter (41 nm) with a narrow distribution were also achieved. The particles still showed excellent dispersity and stability even after spray-dry or freeze-dry process, high temperature sterilized conditions and long-term storage. The nanoparticles could quickly capture iron ions in bulk solution which was confirmed by ITC results, and the bioactive iron of CS015 was greatly decreased (0.54±0.05 mg/L) compared to commercial available Ferumoxytol, Iron Sucrose and VSOP. Free iron ions release was 1120 times lower than iron toxic concentration. An excellent biocompatability of CS015 with no obvious cytotoxicity and low risk of hypersensitivity has been manifested by cytotoxicity experiments and passive cutaneous anaphylaxis test. T1 and T2-weighted MRI contrast effects both in vitro and in vivo had also been verified which made CS015 a potential dual MRI contrast agent. Furthermore, theoretically calculated conformation was speculated and all the advantages mentioned above were benefited from the three dimensional brush- like texture of CS015. Therefore, these merits endow CS015 nanoplatform a high potential in diagnostic applications as a MRI contrast agent.

Introduction
Magnetic nanoparticles (MNPs) especially superparamagnetic nanoparticles have been extensively investigated as a magnetic resonance imaging (MRI) contrast agent due to their enhanced magnetic relaxivity, adjustable bio-distribution and outstanding biocompatibility.1-3 These particles all consist of a nano-sized magnetic core that affects the magnetic property and a functional coating on the surface as the interaction interface of MNPs with bio-systems.4 Both magnetic cores and coating materials need to be carefully designed in order to meet the requirements of bio-distribution and pharmacokinetics in vivo.5 Hence, the control of some key parameters that correlated with the physicochemical properties of particles is very important. Hydrodynamic size (dH) is one of the most important factors to impact bio- distribution kinetics as MNPs may quickly accumulate in liver and spleen through macrophage phagocytosis when dH﹥100 nm while likely be eliminated by kidneys with dH﹤10-15 nm.6,7 Therefore, it is possible to prolong the blood half-life timeand improve the access of MNPs to other organs such as lymph node, brain or tumors by tuning the hydrodynamic diameter of MNPs within 10-100 nm8, 9.Core size is also a vital factor that determines the saturation magnetization and direct T1, T2 and T * when used as a MRI contrast agent.10 A variety of molecules have been used as the coating agents such as natural materials (e.g., dextran, starch) and synthetic polymers (e.g., PEG, co-polymers) to increase the blood circulation time and enhance the stability of particles.11-14

Furthermore, stability of MNPs also plays an essential role in clearance used as a MRI contrast agent can image different tissues include liver, blood vessels and lymph node, etc.18-21In the previous investigations, two kinds of magnetic contrast agent have been classified, one is superparamagnetic iron oxides (SPIOs) which have a hydrodynamic size above 50 nm and are usually retained by reticuloendothelial system (RES), while the other one is ultra-small SPIOs (USPIOs) that tend to escape RES system and phagocytosis by deep macrophagic portion.19 Compared with SPIOs, USPIOs have a smaller dH of ~20-30 nm and exhibit a longer circulation time.22 In fact, many kinds of formula have been tried to develop USPIOs with low toxicity and high efficacy,23 among which, Ferucarbotran (Resovist®, Bayer Helthcare), Ferumoxide (Feridex®, Advanced Magnetics), Ferumoxtran-10 (Combidex®, AMAG Pharma), Feruglose (Clariscan®, Nycomed) and VSOP C184 have been extensively studied in this area. All the particles comprised an iron oxide core and a coating layer of dextran, carboxyl dextran or citrate acid, etc. Unfortunately, most of the intravenous iron agents have been withdrawn from clinic trials due to safety concerns and formula efficiency.24, 25 Bioactive iron that exists in all intravenous iron preparations may be an essential trigger that induces the uncommonly but seriously adverse drug events.26-28 Hypersensitivity reaction (HSR) that has been found in most iron agents29-31 has become a crucial concern for the security of intravenous iron caused by free iron. Both in vivo and in vitro studies have shown that bioactive iron contributes to microbial growth and leucocyte function.32 Furthermore, biologically active or free iron can react with hydrogen oxide peroxide and oxygen produced by mitochondria resulting in highly reactive oxygen species (ROS)33-35 via Fenton reaction. ROS can aggrandize the risk of lipid radicals, contributing to endothelial dysfunction and atherogenesis.36, 37 Hence, minimize the bioactive iron release is imperative to intravenous iron formulations.

In addition, immVieuwnAortgicelenOicnliitnye due to IgG-mediated anaphylaxis has shoDwOnI: 1a0r.1e0la39ti/oCn9sThBi0p0w00it2hJ high molecule weight dextran.38 The other important factor for the iron formulation is colloid stability. The nanoparticles should disperse well in normal saline and blood to avoid particle aggregations. There should be a strong chelation interaction between coating materials and surface iron atoms that avert the detachment of molecules from particle surface, especially when the particles undergo high temperature sterilizing.To solve these problems, a new kind of USPIO, that is CS015 (C stands for carboxyl coatings and S represents superparamagnetic), has been developed in this study. By using a reported microwave assistant polyol process developed by our group,39 it is aimed to design and synthesize CS015 with ultrahigh density of carboxyl groups that can endow CS015 with super stability and lower probability of hypersensitivity. Physicochemical properties, stability, safety and magnetic properties were mainly studied in this work. The particles of CS015 show an excellent stability in varies conditions and even for the long-term evaluation. In addition, safety concerns were explored through bioactive iron release, cell toxicity experiments in vitro and passive cutaneous anaphylaxis test in vivo. Superparamagnetic property and r2/r1 ratio were characterized to evaluate the potential of CS015 as a dual MRI contrast agent. Finally, the conformation structure of the coated layers on particles was deduced to explain the outstanding stability and safety outcomes.

Results and discussion
Characterization of CS015The morphology of CS015 has been characterized by TEM and Fig. 1 Morphology and crystal structure characterization of CS015: (a) TEM images. (b) Size distribution counted from a. (c) HRTEM, insert is SAED pattern. (d) XRD patterns of CS015 with a scanning rate 2°/min and 2θ range from 25° to 70°.Fig. 2 Surface coating characterization of CS015: (a, b) FTIR spectra for blank PAA, neural-PAA and CS015. (c) TGA weight loss of bare Fe3O4, CS015 and PAA under 800 °C with a heating rate of 10°C/min. high resolution TEM (HRTEM). As shown in Fig. 1a, the particles are mono-dispersed on the copper grid even at dense area. It could be clearly observed that there exists obvious gaps between all closely packed particles and the average width of gaps measured in TEM is 0.99 ± 0.09 nm, which is in accordance with the length of dried PAA. Hence, the synthesized CS015 particles exhibit good monodispersity and without any aggregations. As shown in Fig. 1b, the average size of CS015 is 5.1 ± 1.0 nm with a narrow size distribution. Moreover, the HRTEM image (Fig. 1c) reveals well-defined lattice planes with interplanar distances of 2.533 Å and 2.975 Å, which stand for (311) and (220) planes of Fe3O4, respectively.40 The insert in Fig. 1c represents the electron diffraction patterns of CS015 and reflects clear rings, indicating that the solid core own crystalline structures. Furthermore, interplanar spacing (d) is calculated using d = S/(R×n), where S represents the length of scale in the picture, R is the distance from the center to the rings, n stands for the number of reciprocal space scale (here n = 5). The calculation results are summarized in Table S1.

Compared with JCPDS card No.39-1346 (Fe3O4) and No.19-0629 (Fe2O3), the solid core is referred as Fe3O4. In order to further analyze the crystal structure of nanoparticles, XRD pattern was detected and the result is presented in Fig. 1d. Position and relative intensity of all diffraction peaks at 30.06 (200), 35.45 (311), 42.94 (300), 53.54 (422), 56.96 (511), 62.59(440) of the nanoparticles are matched well with magnetitefrom the JCPDS card (No.39-1346). Despite the magnetite peaks, there were no other peaks observed in the diffraction pattern of CS015. The average size calculated by Debye- Scherrer’s equation is 5.3 nm, which is in accordance with TEM results.The most significant formula modification for CS015 is utilization of small molecular PAA as surface coating material. Characterizations of FTIR and TGA have been applied to verify the components and contents of PAA on the surface of particles. Fig. 2a shows the FTIR spectra of CS015 with PAA and neutralized PAA as controls in full wavenumber, and Fig. 2b is the enlargement from 400 to 1800 cm-1. All the samples show broad absorption bands at 3356, 2935 cm-1, assigned to the – OH stretching vibration, the asymmetric and symmetric stretchof -CH2, respectively.41 For the blank PAA, 1697 cm-1 is a characteristic peak, which represents -C=O stretching of carboxylic groups.42 However, the relative intensity of 1697 cm-1 peak in neutralized PAA and CS015 is decreased and there appears a new band at 1552 cm-1, which should be attributed to the asymmetric stretching vibration of -COO groups.43, 44 What’ more, the shift of peak at 1697 cm-1 to 1717 cm-1 in CS015 indicates the possible formation of some ester linkage.

The peak of 1412 cm-1 could be assigned to the symmetrical-COO stretching, which shifts to 1400/1357 cm-1 in neutral- PAA/CS015, manifesting the interactions between PAA functional group and iron oxide surface. According to literatures, it is possible to deduce the bidentate conformation using the following equation: Δν = (νasym – νsym). For neutralized PAA, Δν is 152 cm-1 which is relevant to carboxylate salt of – COONa. For CS015, Δν is 155 cm-1 which is very similar to that of neutralized PAA, illustrating that the interactions between PAA and Fe3O4 surface are mainly classified as Fig. 3 Dispersity of CS015 records by DLS: (a) after spray drying, freeze drying, and high temperature sterilizing (HTS). (b) Under 0.067 M pH 7.4 PBS and SBF after 4 weeks (c) keep in water for 1 year. chemisorption.46, 47 The weak band at 1447 cm-1 is assigned to-CH2 scissoring vibration and it remains unchanged in all samples, demonstrating that the conformation of PAA on Fe3O4 surface keeps more in loops than in trains.44 These results are in accordance with the calculated conformation in supporting information. Additionally, the peak at 561 cm-1 is ascribed to Fe-O stretching vibration. TGA curves of CS015 with bare Fe3O4 and PAA as controls are exhibited in Fig. 2c, and the weight loss of 45.94% quantifies the organic coating of PAA. The first stage of weight loss before 150 °C is defined as the evaporation of physically absorbed water while 150 °C – 700 °C is attributed to organic decomposition.48 13CNMR was also applied to detect the surface composition (Fig. S2), in which 183 ppm represents C atom in -C=O and illustrates the exist of small amount of ester.49 In brief, PAA is successfully coated on the surface of Fe3O4 cores.In order to explore the binding state of PAA on the surface of nanoparticles, total content of free COOH groups was measured by using conductance titration analysis with sodium hydroxide.

The amount of carboxyl group was calculated from titration curve (shown in supporting material Fig. S1) with a result of 9.0 ± 0.1 mmol/g Fe for CS015. As showed in supporting, there were 2727 COOH groups on one particle with a grafting density 2.35 COOH/nm2 (see conversion in experimental section and detailed calculation process in supporting information), which is extremely high compared to ordinary coating strategies as atom transfer radical polymerization (ATRP) with 0.5-0.6 chains/nm2, reversible deactivation radical polymerization (RDRP) with0.4chains/nm2 and surface-initiated RAFT polymerization with1.43chains/nm2.50-52 This high coating density is assumed to derive from microwave assisted polyol method using in our work as it can speed up reaction kinetics and reduce reaction time compared to conventional convective and conductive heating techniques.53-55 Physicochemical properties of CS015 are summarized in Table 1. The most significant characteristics of CS015 are their ultra-small size, narrow size distribution and outstanding high carboxyl group coating density.Colloidal stability of CS015 The colloidal stability as well as dispersity of particles in a series of different conditions are studied and recorded by DLS. As depict in Fig. 3a, DLS curves of re-dissolved aqueous suspension after spray dried, freeze dred and high temperature sterilizing (HTS) are shown. In Fig. 3b, it depicts the stability of CS015 under 0.067 M pH 7.4 PBS and SBF after 4 weeks as well as (c) kept in water for 1 year. The relative images are also shown in Fig. 3. As can be seen, CS015 is dispersed well after re-dissolving the spray dried and freeze dried samples for a few days. The DLS curves of re-dissolved nanoparticles almost keep unchanged compared to each other, illustrating the excellent stability of CS015 during drying processes. What’s more, there is no difference in size distributions after sterilization at 120 °C for 30 min, indicating that the chelating between PAA and particles are strong enough to avoid PAA detachment from the surface. After dispersed in 0.067 M pH 7.4 PBS and SBF for 1 month, the peak intensity get smaller in SBF because there are a few divalent ions in SBF which can coordinate with PAA and make PAA chains collapse. In the meanwhile, a little right shift is appeared in PBS, this may induce by polyelectrolyte effect that the weakly alkaline solution made PAA chains straighter.56 Long term stability also studies as shown in Fig. 3c, no obvious difference is found after 1 year’s storage of CS015 under room temperature.Free iron prevention of CS015Bioactive iron may trigger uncommon and serious adverse drug events for IV iron formulations.

Nanoparticles that can restrain iron ions release and chelate free iron ions will be a crucial factor to improve the safety for clinical use. The main consideration for the molecule design of CS015 is trying to avert iron release to bulk solution through ultrahigh carboxyl group coating density. As most of ions in vivo are divalent ions, common divalent ions (Fe2+, Ca2+, Mg2+) are chosen. In order to investigate whether there is absorption effect between CS015 and ions, isothermal titration calorimetry (ITC) is utilized. From the thermal effect, ITC can give thermodynamic parameters of binding stoichiometry (n), binding constant (K), changes of entropy (Δ S) and enthalpy (ΔH). Peaks above zero in thermo-Fig. 4 Raw ITC thermo-gram (up) and corresponding Wiseman diagram (down) of the interaction between 1.5 mM COOH in CS015 and 4 mM (a) FeCl2, (b) CaCl2 and (c) MgCl2. gram correspond to endothermic reaction and below zero represents exothermic heat effect. Corresponding Wiseman diagram is integrated from the peak area of thermo-gram and are fitted by nonlinear least-squares. As shown in Fig. 4, strong interactions of CS015 with ions are clearly observed from the sharp endothermic peaks and the binding site n was 0.237, 0.236, 0.208 for Fe2+, Ca2+, Mg2+, respectively. This indicates that there are about 5 COO- combined with 1 ion. The association constants (K) are very high for all kinds of ions. Free energy change can be calculated using equation ΔG =ΔH – TΔS where T was cell temperature, and the results were -7.33 kcal/mol for Fe2+, -7.30 kcal/mol for Ca2+ and -6.87 kcal/mol for Mg2+, which means it is a spontaneous process between CS015 and ions. There are obvious exothermic peaks during the reaction between Fe2+ and CS015.

This phenomenon is caused by the hydroxylation of Fe2+ that form [Fe(OH)(OH2)5]+ in alkaline CS015 suspension (see supporting Fig. S3).57 The reason that CS015 absorbs ions is due to the large amount of COO- on the particle surface that COO- can chelate with ions. Therefore, the ability to absorb Fe2+ in bulk solution of CS015 may decrease oxidative stress and greatly enhance security both in vitro and in vivo. The high affinity with iron ions also imply the stable chelating of PAA with surface Fe atoms of iron oxide particles.Biological free iron of CS015 before (Un-Ster) and after sterilization (Ster) are detected. To further prove the absorption ability of CS015, free iron concentration after adding 50 mg/L FeCl2 directly to CS015 samples are verified (SAM, standard addition method). Free iron ions release is conducted by keeping CS015 (without free ions) at 70 °C for 15 days. Three different coating materials are used as control: Ferumoxytol (Feru, dextran), Iron Sucrose (IS, sucrose) and very small superparamagnetic iron oxide nanoparticles coated with citrate acid (VSOP). The results reveale in Fig. 5a show that bioactive iron concentration of CS015 before (0.54±0.05 mg/L) and after sterilization (0.53±0.02 mg/L) is similar to each other stating sterilization almost have no effect on free iron content. The detect concentration of iron ions for SAM group is 0.75 ± 0.035 mg/L, which is just a little higher compared with un-sterilization and sterilization samples but much lower than the amount that add (50 mg/L). These results indicate the good property of CS015 in restricting ions to bulk solution. Free iron ions release (Sec) is 0.05 ± 0.008 mg/L which is 1120 times lower than clearly toxic concentration (1 mM) implying a lower toxicity as it may induce ROS and injure human body.58-60 Compared to other coating materials: Feru (0.86±0.046 mg/L), IS (2.12±0.028 mg/L) and VSOP (10.05 ±0.54 mg/L), the lowest free iron is showed in PAA coated particles. All in all, CS015 not only can quickly capture iron ions in bulk solution but also can prevent it diffuse to bulk solution, which illustrates advantages in minimizing probability of hypersensitivity.Cell viability present in Fig. 5b is used to assess the toxicity of magnetic nanoparticles by using human lung adenocarcinoma cell line H1299 as models with CCK-8 method. The viability of incubated cells are almost stable with the increasing concentration of Fe (50-500 μg·mL-1) which demonstrate the low cytotoxicity of all kinds of samples.

There is slightly decrease in Ferumoxytol at high concentration, but the viability is still above 93%. Percentage above 100% may be contribute by cell growth.61Passive cutaneous anaphylaxis test is utilized to further evaluate the security of CS015. NaCl and hen ovalbumin injection are used for negative and positive groups, respectively. CS015, Ferumoxytol and IS are used for sample group. As described in Fig. 5c, the injection site of serum onFig. 5 (a) Free iron release of CS015 before sterilization (Un-Ster), after sterilization (Ster) and standard addition method (SAM) of aid 50 mg/L FeCl2 in 2 mL 2 mg/mL Fe samples, iron release after 15 days at 70 ℃ (Sec), free ion content of Ferumoxytol, iron sucrose and VSOP. (b) Viability of H1299 cells of three samples with different Fe concentration after incubated for 24 h. (c) Passive cutaneous anaphylaxis in mice-rat.: negative control group, positive control group, CS015 sample group, Ferumoxytol sample group and iron sucrose group. dorsum of rat as well as passive cutaneous anaphylaxis results of negative, positive and sample groups are showed. Blue plaques in negative and sample groups barely appear while it is obvious in positive group. These results illustrate that even the bioactive iron release for IS is a little higher, no anaphylaxis reaction was found, which further manifest the high security of CS015 and its potential application in MRI. As shown in Fig. 6a, magnetization vs applied magnetic field is determined at 298 K for CS015 under ± 2T. Insert is corresponding magnetization curves at low field. CS015 performes no remanence and coercivity which indicate its superparamagnetic property.62 The saturated magnetization of Fig. 6 (a) Magnetization curves of CS015 at room temperature under ± 2T, inset was corresponding magnetization curves at low magnetic field. (b) Evolution of 1/T1 and 1/T2 with different concentration of Fe ions and the corresponding relaxation rates were shown inside. (c) In vitro T1- and T2-weighted images recorded on a 3T scanner.Fig. 7 T1 -weighted MRA images of rabbit vasculatures after injection of 150μmol Fe per kg CS015 (a, b). T2 -weighted MRI images of rabbit popliteal lymph node before (c) and after (d) injection of 150 μmol Fe per kg CS015. weighted MR images are obtained using a seVrieiwesArtioclfe Oinrolinne concentration (0, 1.0, 1.5, 2.0, 2.5, 3.0,D4O.I0: 10m.1M03)9/uCn9dTBe0r0a0032TJclinical scanner. As reveal in Fig. 6c, a bright signal enhancement is displayed in the T1-weighted MR image with increasing iron concentration from 0 to 4 mM. Similarly, in the T2-weighted image CS015 also exhibit a signal decrease with iron aggrandizing. These results illustrate CS015 could be a potential T1 as well as T2 contrast agent.

T1 contrast effect of CS015 is evaluated in vivo for blood vessel imaging using rabbit. The injection dose of CS015 is 150 μmol Fe per kg and images are acquired subsequently. In Fig. 7a and b, the coronal scanning MR images of rabbit blood vessels are imaged. The arteries are clearly visualized through 3D-FSPGR-TOF sequence. What’s more, even the small vessels in renal arteries and ramifications around pulmonary artery can be observed successfully, which fully reveal the high resolution after injected CS015. Furthermore, T2-weighted MRI images of rabbit popliteal lymph node are obtained after injection CS015 for 24 h with a dosage of 150 μmol Fe per kg. As shown in Fig. 7c and d, in comparison with pre-contrast images, the lymph node signal intensity is obviously decreased with a percentage of 130%. These results exhibit the great potential of CS015 as a dual functional contrast agent.As discussed above, CS015 has ultrahigh density of carboxyl group on the particles surface and shows outstanding dispersity and stability. The particles can also absorb iron ions in bulk solution with greatly decrease bioactive iron. Lowcytotoxicity has been verified and CS015 exhibit highly security the particles was 59.16 emu/g and it was lower than the bulkmagnetite (92 emu/g)63. This may be due to small size particles as it has been reported that Ms is linearly correlated with particle size but no definite evidence shows that the content of coating materials is proportional to saturation magnetization.64, 65 The plots in Fig. 6b represent relaxation rates Ri=1/Ti vs Fe concentration (0.2, 0.6, 1.0, 1.5, 2.0 mM). The slopes are the relaxivities r1 and r2, and appear in units of mM-1 s-1.

Corresponding longitudinal/transverse relaxivities r1 and r2 of CS015 are 10.52 mM-1 s-1 and 38.97 mM-1 s-1, which are respectively three times and eight times of Gd-DTPA agent (r1 = 3.8 mM-1 s-1, r2 = 4.2 mM-1 s-1) measured under the same conditions.66 CS015 also has a competitive r1 value and r2/r1 ratio in contrast to SHU 555C (r1 = 10.7 mM-1 s-1, r2 = 38 mM-1 s-1, r2/r1 = 3.6).67 The enhancement in longitudinal relaxation is due to the small size of CS015 which decrease magnetism moment, increase surface ferric ion and finally aggrandize T1 effect.68 High r1 as well as low r2/r1 ratio make CS015 an ideal candidate for T1 and T2 dual contrast agent.69-71 To further investigate the underlying dual-contrast agent, T1- and T2- in passive cutaneous anaphylaxis. All these merits may derive from the unique structure of CS015 especially its ultrahigh density of COOH. Hence, the conformation of particles is studied with the calculation process presents in Supporting Information with results exhibit in Table 2. As shown in Fig. 8, the final texture of CS015 is speculated to be densely packedbrush-like in whole look with a mixture structure of loops and tails in the inner part, which is in accordance with FTIR results. Almost all the Fe atoms on the particles surface are chelated with COOH and finally formed this brush-like structure. For each PAA chain, the number of chelated COOH with surface Fe atoms is about 3 while the whole average number of COOH groups is 14.

This highly biding site not only suppress PAA detachment from particles even at high sterilizing temperature but also benefit to the stability of particles.72 Profited by this “3D” texture, it provides much more space for free COOH to absorb ions as a result greatly decrease iron ions release and finally enhance security. The conformation of densely pack brush-like structure may come from the special microwave- assistant polyol process, during which Fe2+ and Fe3+ maybe fully coordinate with PAA when adding NaOH.Ferric chloride hexahydrate (FeCl3·6H2O) and Ferrous chloride (FeCl2) are purchased from Shanghai Macklin Biochemical Co., Ltd. Sodium hydroxide (NaOH), diethylene glycol (DEG), sodium chloride (NaCl), citrate acid, ammonium hydroxide, sodium dihydrogen phosphate (NaH2PO4), dipotassium phosphate (K2HPO4), hydrogen chloride (HCl), nitric acid (HNO3), sodium hydrogen carbonate (NaHCO3), magnesium chloride hexahydrate (MgCl2·6H2O), calcium chloride dihydrate (CaCl2·2H2O), sodium sulfate (Na2SO4) are bought from Sinopharm Chemical Reagent Co., Ltd. Polyacrylic acid (PAA, Mw 1000), HEPES (﹥99%) is purchased from J&K Chemical. Cell counting kit-8 (CCK-8) and dialysis membrane (MWCO: 100-500 D) are bought from Kanwin Biotech Co., Ltd. DMEM supplement is purchased from ThermalFisher scientific. All the reagents are used without further purification. Millipore Milli- Q water is used in all experiments. Citrate acid coated nanoparticles (VSOP) are synthesized using former work by others.73Synthesis CS015PAA coated magnetic nanoparticles are synthesized using microwave-assistant polyol process in our group39. Briefly, 8 g NaOH are dissolved in 80 mL DEG at 50 °C for 4 h and stored at 72 °C for further use. Then, 6.921 g PAA, 7.8 g FeCl3 are added into 360 mL DEG. The mixture is transferred into a flask and heated to 220 °C with vigorous stirring at about 270 rpm and keep for 5 min. Followed by injecting the NaOH rapidly into the flask and maintain 10 min at 220 °C.

The final product is ultrafiltration with Mw 100,000 spiral membrane until the conductivity below 10.Physicochemical characterizationsDynamic light scattering (DLS) is used to detect the hydrodynamic size and surface charge of particles and performed by a particle sizing system of Zetasize Nano ZSP with a scattering angle of 90°. The sample is prepared by 2 mL water contained 20 μL CS015. Transmission electron microscopy (TEM) is applied to analyze morphology, size and structure of CS015. TEM images and electroVniewdAirftficrlae cOtniloinne patterns are obtained on a HEOL-2010DwOIi:t1h0.a10n39a/cCc9eTlBe0r0a0t0in2gJ voltage of 200 KV. Samples that diluted with water are drop- cast on carbon-coated copper grids. The average particle size is measured from more than 300 particles in the TEM images. Powder X-ray diffraction (XRD) is performed with a Rigaku Dmax-r C X-ray diffractometer at 40 kV using Cu Kα radiation (λ= 1.540 Å) and 100 mA for further determine the crystal structures of particles. The scanning speed is 2°/min with a scanning range between 20° and 70°.Fourier transform infrared spectra (FTIR) are collected on a TENSOR 27 (BRUCK Platinum ATR) instrument to verify the component of coating. Powder samples detect immediately. PAA is neutralized with 0.1 M NaOH and freeze dried. The content of organic part is performed by a thermal gravimetric analyzer (TG 209 F1, NETZSCH) with a heating rate 10 °C/min from 35 °C to 800 °C in air flow. Dissociative carboxyl is measured using conductance titration (T50, Mettler Toledo) with 0.1 M NaOH. Sample about 200 μL is diluted to 50 mL with water, adding 800 μL 0.1 M HCl to the titration cup and titrated. C13-NMR is processed on a 600 MHz nuclear magnetic (Advance III600 MHz) with a magnetic field 14.09 Tesla. Samples (1 mL about 20 mg/mL) are splitting with HCl and the supernate is neutralized with NaOH (named a) and freeze- drying. Some of neutralized supernate is dialyzed and freeze- drying (named b). Fe concentration: 1mL HCl/HNO3 (v/v: 3/1) isadded to 200 μL sample, keep in 60 °C water bath for 1 h and dilut 10000 times for atom absorbance spectroscopy (AAS) detection. COOH content is calculated as follows: 2702/(1000/72)/81.6 = 2.35 chains/nm2 (1000 for PAA Mw, 72 for acrylic acid molecular weight, 81.6 was surface area of 5.1 nm Fe3O4 core).Colloidal stability assaysSpry drying (XINW-6000, Xinweng) is used to dry CS015 at 200 °C. Freeze drying (BETA 1-8, Christ) is used to freeze drying CS015.Safety experimentsIsothermal Titration Calorimetry (ITC, ITC200, Malvern) is utilized to confirm the adsorption process and integrating ability between CS015 and ions. The concentration of COOH on the nanoparticles is diluted to 1.5 mM in cell and 4 mM FeCl2, CaCl2, MgCl2 in the syringe with a titration injection 2 μL per drop under 25 °C, respectively.

Acidic condition of CS015 is adjusted to pH 5.5 with final concentration of COOH 1.5 mM in the cell and 2 mM FeCl2 in the syringe. Free iron content samples are prepared by centrifuging 2 mL 2 mg/mL Fe concentration of CS015 before and after sterilization (XFH- 50CA, Zhejiang Xinfeng), Ferumoxytol, iron surcrose (IS) and VSOP 10 min at 6000 rpm in ultrafiltration tube (MWCO 3000D). Standard addition method (SAD) is applied to investigate the adsorption of Fe2+. FeCl2 is added to 2 mL 2 mg/mL CS015 with a final concentration of 50 mg/L then centrifuge at the same conditions. Free ions release conduct by removing free irons using ultra-filtration CS015 for 3 times and keep the sample at 70 °C for 15 days, then ultrafiltration 2 mL 2 mg/mL CS015 again. Soon afterwards, the ultra-filtrate is detected by AAS.Cell viability CCK-8 assay: Human lung adenocarcinoma cell line H1299 are seeded in a 96-well plate with 100 μL culture DMEM medium contained 10% fetal bovine serum, 100 kU/L penicillin and 100 mg/L streptomycin. The cells are incubated for 24 h at 37 °C in a 5% CO2 incubator using. Subsequently, 10 μL CS015, Ferumoxytol and IS with different Fe concentration (0.55, 1.1, 2.2, 3.3, 5.5 mg/mL) are addedinto each well with a final Fe concentration 50, 100, 200, 300, 500 mg/L. Every sample is prepared for 5 plates and each concentration was set 5 wells, incubate for 24 h. Then, samples are removed and wash with PBS gently. 100 μL culture medium containe 10% cell counting kit-8 (CCK-8) is added to each well and incubate for another 1.5 h. The optical density (OD) value is detected using an enzyme-linked immunsorbent assay plate reader at wavelength 450 nm (Spectra Max i3x, Molecular device). Cell viability is calculated by the ratio of OD values from experiment group, control group and blank group.Passive cutaneous anaphylaxis test: Briefly, negative control group (NaCl injection), positive group (hen ovalbumin) and sample group (CS015, Ferumoxytol, IS) are designed. Firstly, 30 mice are selected and divided into 5 groups and are subcutaneous injected with NaCl injection, hen ovalbumin, CS015, Ferumoxytol and IS, respectively.

After final active sensitization, draw blood and extract serum for further use. Then, 15 rats random are separated into 5 parts, chose 6 sites on dorsum of each rat and inject the corresponding serum intradermic. One day later, 51 mg/g sample is given to negative group intravenously and the other two are the same dosage with active sensitization. Followed by injecting 0.5 mL 20 mg/mL evans blue solution. Cut dorsum and observed the blue plaques.Magnetic propertiesIn vitro: Vibrating sample magnetometer (VSM) PPMS-9T (EC-II) is used from -2 T to +2 T with a step rate of 100 Oe/s at 298 K. The sample is freeze-dried to solid. Relaxation times experiments are recorded using NMR analyzer Bruker mq 60 (60 MHz, 1.41 T) at 37 °C. The T1,2 relaxation rate (r1, r2) are calculated by fitting inverse relaxation time (1/T1,2) as a function of iron concentrations. MR phantom study images are obtained on a GE 3T Discovery MR750w Medical Systems at ambient temperature (~25 °C). T1-weighted image is achieved using fast recovery fast spin echo pulse sequence with TR = 500 ms, TE = 8.4 ms, FOV = 20 × 20 mm2, data matrix = 224 × 192, slice thickness = 3 mm, NEX = 6. T2-weighted image is acquired using fast recovery fast spin echo pulse sequence with TR = 1000 ms, TE = 16.8 ms, FOV = 20 × 20 mm2, data matrix = 224 × 192, slice thickness = 3 mm, NEX = 6. Samples are prepared by diluting CS015 to different Fe concentration with water (0, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 mM) and placed on a tube shelf.To evaluate T1 and T2 contrast effect of CS015 in vivo, magnetic resonance angiography (MRA) and MRI at popliteal lymph node images are scanned using rabbits. Animal tests are permitted by the animal care and use committee of Shanghai Jiao Tong University. MRI is applied on a 3T ViGewEArtMicleeOdniclinael Systems (discovery MR750w) using a GEMDOFIl:e1x0.1C0o3i9l /1C69-TmB0.0M00R2AJ with 3D Fast Spoiled Gradient Echo (3D-FSPGR-TOF) sequence (TR = 5.4 ms, TE = 1.6 ms, filp angle = 30°, FOV = 20 cm2, slice thickness = 0.8 mm) is obtained. Popliteal limph node images with 3D Gradient Echo (3D-GRE) sequence (T2WI: TR = 8.9 ms, TE = 3.2 ms, filp angle = 30°, FOV = 10 × 7.5 cm2, slice thickness= 2 mm) is applied. Rabbits are narcotized using pentobarbital sodium with the dose of 45 mg/kg through ear vein injection. The dose of injected CS015 is 150 μmol Fe per kg and images are acquired immediately after injection at abdominal aorta and popliteal lymph node after 24 h.All data showed are the average ±SD of experiments repeated for three or more times. If imparity were statistically great, a Student’s t-test is needed to check. A p value of <0.05 is preferred to certificate differences between groups. Conclusions To summarize our work, low molecular weight PAA-coated Fe3O4 nanoparticles (CS015) are synthesized successfully using microwave assistant polyol method. The constitution of particle is analyzed using many instruments to confirm the 5.1 nm Fe3O4 core and high density of low molecular weight PAA coating. Dispersity and stability of nanoparticles are excellent at physiological condition as well as after long storage period. From ITC results, CS015 can capture iron ions quickly in bulk solution. Bioactive iron is the lowest compared with other coating materials. CS015 can also restrict iron ions in inner part and low iron release may greatly reduce ROS. Cytotoxicity experiments and passive cutaneous anaphylaxis test exhibit low cytotoxicity and less risk of hypersensitivity of CS015. The ratio of r2/r1 is 3.7 that make CS015 a potential dual functional MRI contrast agent which further verify by T1 and T2–weighted effects in vitro and in vivo with animal tests. According to theoretical calculation, there are 3 COOH on each PAA chain binding with surface Fe atoms and the conformation is deduced to be brush-like. Due to this special “three dimensional” Iron sucrose texture, CS015 reveals a series of merits that has been mentioned above. Altogether, CS015 shows a great promise in MRI contrast agent and worth further clinical development.