Strategy to prevent cardiac toxicity induced by polyacrylic acid decorated iron MRI contrast agent and investigation of its mechanism

Biomaterials 222 (2019) 119442

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Strategy to prevent cardiac toxicity induced by polyacrylic acid decorated iron MRI contrast agent and investigation of its mechanism

T

Hao Fua,1, Chongchong Miaoa,1, Yuanpeng Ruib, Fenglin Hua, Ming Shena, Hong Xua, Chunfu Zhanga, Yi Dongc, Wenping Wangc, Hongchen Gua,∗∗, Yourong Duana,∗ a

State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China Department of Radiology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China c Department of Ultrasound, Zhongshan Hospital, Fudan University, Shanghai, China b

ARTICLE INFO

ABSTRACT

Keywords: Ion balance Cardiac toxicity Polyacrylic acid Iron oxide nanoparticles

Polyelectrolyte modified iron oxide nanoparticles have great potential applications for clinical magnetic resonance imaging (MRI) and anemia treatments, however, possible associated heart toxicity is rarely reported. Here, polyacrylic acid (PAA)-coated Fe3O4 nanoparticles (PION) were synthesized and lethal reactions appeared when it was applied in vivo. The investigation of underlying mechanism showed that PION could break electrolyte balance and further resulted in serious heart failure, which was observed under color doppler ultrasound and dynamic vector blood flow technique. The results demonstrated that PION had a strong absorption tendency for divalent ions and the maximum tolerated dose (MTD) was lower than 100 mg/kg. From electrocardiography (ECG), PION presented an obvious impact on CaV1.2 ion channel, which leading to fatal arrhythmia. An appropriate solution for preventing this deadly effect was pre-chelation Ca2+ (n (Ca): n (COOH) = 3: 8) to PION (PION-Ca), which displayed much higher cardiac and electrophysiological safety when sealing the binding point of divalent cation ions with PAA. The injection in Beagle dogs further confirmed the safety of PION-Ca. This study explored the mechanism and offered a solution for cardiac toxicity induced by PAA-coated nanoparticles, which guides for enhancing the safety of such polyelectrolyte decorated nanoparticles and provides assurance for clinical applications.

1. Introduction Magnetic resonance imaging (MRI) has been widely utilized since the 1980s and many contrast agents have been extensively used in clinical settings. To increase tissue contrast, paramagnetic materials have been used either as T1 or T2 MRI contrast agents [1,2]. For T1 contrast agent, gadolinium-based contrast agents (GBCAs) are clinical widely utilized due to their high magnetic moment and ease to excrete within several days after administering the GBCAs [3]. However, GBCAs still have some drawbacks. For example, some GBCAs have been found to show long-term toxicity with nephrogenic systemic fibrosis (NSF) in patients with significant renal impairment [4]. It owns a short blood half-life time due to the small size that enables passive glomerular filtration. A symptomatic disease process is observed in individuals with normal renal function caused by gadolinium deposition, which includes central or peripheral pain, headache, and bone pain,

further, 98% of patients have persistent gadolinium detected in urine samples [5]. Because of the disadvantages of gadolinium, T2 contrast agent, especially iron oxide nanoparticles (IONPs), have attracted the attention of scientists and have been extensively studied in recent years [6–8]. Compared to GBCAs, IONPs are biocompatible, biodegradable and nontoxic [9]. According to the design of different coating materials or sizes of IONPs, they can be used in various tissues, such as the liver, cancerous tissues, blood vessels [10–13]. Additionally, IONPs can be used both for T1 and T2 contrast agents [14], and they were approved by the FDA for clinical application in the 1980s and have subsequently been used commercially. Feraheme®, though not used as a contrast agent, is still utilized with clinical intravenous iron deficiency due to the reabsorption of iron through normal iron metabolic pathways [15], which demonstrates the great potential of INOPs applied as an MRI contrast agent. Over the past years, many superparamagnetism iron oxide (SPIO)

Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Gu), [email protected] (Y. Duan). 1 These authors contributed equally to this work. ∗

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https://doi.org/10.1016/j.biomaterials.2019.119442 Received 23 April 2019; Received in revised form 30 July 2019; Accepted 20 August 2019 Available online 22 August 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.

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was solved by pre-chelation Ca2+ in PION (PION-Ca) with relevant process shown in Scheme 1. The basic physicochemical properties and interactions between PION/PION-Ca and ions in vivo and in vitro were also investigated. The results lay a basic foundation for the development of a PAA-coated MRI contrast agent for use in clinical settings. This new agent can also play a guiding role in the prevention of toxicity of polyelectrolyte decorated nanoparticles.

nanoparticle preparations have been investigated for clinical use, which includes Feridex®, Resovist®, Combidex® and Fereheme® [16]. However, Feridex®, Resovist®, and Combidex® as MRI contrast agents were withdrawn from the market and Fereheme®, was applied for intravenous iron deficiency, received a box warning label by the FDA for possible serious hypersensitivity reactions (HSR) [17,18]. Feridex® and Resovist® had been used for liver imaging, but the former cannot be administered as an intravenous bolus, while there was no significant difference between well-differentiated hepatocellular carcinoma and surrounding normal liver kupffer cells in the latter [19]. Hence, they were withdrawn from the market. Combidex® was utilized for lymph node imaging and clinical development was stopped due to lack of sensitivity and inability to confirm noninferiority specificity [20]. Despite all the challenges depicted above, HSR has been the main factor that affects the safety of iron agents. The symptoms of HSR include dyspnea, chest/back pain, hypo/hypertension, rash, fever, panic, and flushing [21]. As 99% of iron in red cells is recycled, antigenic or allergenic properties have rarely been found for iron. HSR may be induced by bioactive iron as free iron ions can produce highly reactive oxygen species with hydrogen oxide peroxide and oxygen via the Fenton reaction [22,23]. Furthermore, immune complex anaphylaxis resulting from preexisting dextran-reactive antibodies with high molecular weight iron dextran has been demonstrated repeatedly [24]. Hence, one of the investigated points in developing iron agents is to decrease the possibility of HSR occurrence. In a previous work of our group, a new contrast agent, PAA-coated magnetic nanoparticles (PION), was synthesized, and it was shown to possess ultrahigh stability and low free iron ion release with no hypersensitivity and good biocompatibility [25,26]. However, adverse effects have occurred during acute toxicity trials. After a series of experiments, cardiac toxicity was demonstrated with our nanoparticles during intravenous injection. The mechanism that PION uses to induce cardiac toxicity was fully studied in this work and the toxicity problem

2. Materials and methods 2.1. Materials Ferric chloride hexahydrate (FeCl3·6H2O) was purchased from the Shanghai Macklin Biochemical Co., Ltd. Sodium hydroxide (NaOH), diethylene glycol (DEG), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO4), potassium chloride (KCl), dipotassium phosphate (K2HPO4), magnesium chloride hexahydrate (MgCl2·6H2O), 1Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and calcium chloride dihydrate (CaCl2·2H2O) were bought from the Sinopharm Chemical Reagent Co., Ltd. Polyacrylic acid (PAA, MW 1000) was purchased from J&K Chemical. 5-Aminofluorescein (5-AF) was bought from Dalian Meilunbio Co., LTD. Pentobarbital sodium, DMSO, HEPES, potassium gluconate (K-Gluconate), Glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Cisapride and Nifedipine were obtained from Sigma. Fetal bovine serum (FBS), DMEM culture medium and 0.25%-Trypsin-EDTA were bought from Gibco. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was bought from Sigma Aldrich (Shanghai, China). L929 (fibroblast cells) and Ana-1 were obtained from ATCC. Adenosine triphosphate (ATP) was obtained from MP. Barium chloride (CsCl) and barium fluoride (CsF) were obtained from Amresco and Innochem, respectively. hERG HEK-293, NaV1.5 HEK-239 and CaV1.2 CHO cell lines were purchased

Scheme 1. Mechanism of cardiac toxicity induced by PAA decorated Fe3O4 nanoparticles (PION) and the method to reverse this lethal property. After injection of bare PION, the lethal reactions of mice appeared, such as labored breathing, serious cramping and sharp dropping of the heartrate lead to the deaths of the mice. However, when PION-Ca (PION pre-chelation with Ca2+) was injected, nearly all the lethal reactions disappeared from the mice, and almost no deaths of mice were reported. Regarding the mechanism of this phenomenon, a strong interference of ion balance of PION was found and confirmed, which showed the main cause of mice death. 2

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a density of 5 × 104 and incubated with 1 ml DMEM containing 10% FBS. PION-Ca@5-AF (1 mM Fe) were added to each well. After incubation for 2, 8, 12 and 24 h, the L929 cells were washed 3 times with PBS and fixed with 4% Paraformaldehyde and later dyed with DAPI (1 μg/ml) for 10 min. After, the round coverslips were transfer to slides which added with glycerin. Leica TCS SP8 confocal microscope was utilized for in vitro internalization of PION-Ca@5-AF. Mean fluorescence intensity (MFI) of both 5-AF and DAPI were calculated with assistance of Image J software.

from Creacell. The Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (IPSC-CMs) and their culture medium were obtained from Cauliscell. Gd-DTPA was bought from Bayer HealthCare Pharmaceuticals. 2.2. Synthesis of PION and PION-Ca@5-AF PAA decorated magnetic nanoparticles were synthesized using a microwave-assistant polyol process by our group. Briefly, 8 g of NaOH were dissolved in 80 mL of DEG at 50 °C for 4 h in a water bath and stored at 72 °C for further use. Then, 6.921 g of PAA and 7.8 g of FeCl3 were added into 360 mL of DEG and mixed with ultrasound. The mixture was heated to 220 °C with vigorous stirring at 270 rpm and was maintained for 5 min. Then, NaOH was poured rapidly into the flask and maintained for another 10 min at 220 °C. The final product was subjected to ultrafiltration, and the conductivity was below 10. PIONCa@5-AF was synthesized using the protocol of our group [27]. Briefly, the molar ratio of COOH and 5-AF was 5:1, dissolve PION and 5-AF with MEST and DMSO, 37 °C incubated for 1 h, then added EDC incubated for another 3 h, ultrafiltration for 5 times and diluted in water. The excitation wavelength is 488 nm.

2.5. Serum-induced aggregation and storage stability assay Serum stability of PION-Ca was monitored by using relative turbidity [28]. Briefly, PION-Ca (14.13 mg/ml) was added with 30% FBS (v/v) and then incubated at 4 and 37 °C under sterile condition. After diluted for 30 × with FBS, the absorbance of samples was measured at 400 nm, and a corresponding amount of serum alone was used as a reference. The absorbance was measured at 0, 4, 8, 24, 96 and 240 h. Relative turbidity value of 1 indicated that the turbidity of the serumincubated samples was equal to the turbidity of water-incubated samples. The storage stability of PION-Ca was carried out under water condition at 4 and 37 °C in closed EP tubes. The average size of samples was determined by dynamic light scattering (Malvern Zetasizer nano ZS, UK).

2.3. Physicochemical characterizations Transmission electron microscopy (TEM) was utilized to analyze the morphology, size and structure of PION and PION-Ca. TEM image was performed on a HEOL-2010 with an accelerating voltage of 200 KV. Samples were drop-cast on carbon-coated copper grids. The average particle size was measured using more than 300 particles in the TEM images. Powder X-ray diffraction (XRD) was obtained with a Rigaku Dmax-r C X-ray diffractometer at 40 kV using Cu Kα radiation (λ = 1.540 Å) and 100 mA to further determine the crystal structure of the particles. The scanning speed was 2°/min from 20° to 70°. Fourier transform infrared spectra (FTIR) were collected on a Nicolet 6700 instrument to perform the component of coating. Powder samples were ground with KBr and compressed into pellets. Liquid samples were dropped on KBr pellets. A vibrating sample magnetometer (VSM) PPMS-9T (EC-II) was applied from −2 T to +2 T with a step rate of 100 Oe/s at 298 K. The sample was freeze-dried to a solid. Dynamic light scattering (DLS) was utilized to detect the hydrodynamic size and surface charge of the particles, which was performed with a particle sizing system of Malvern Zetasizer nano ZS with a scattering angle of 90°. The samples were prepared with 2 mL water containing 20 μL of PION and PION-Ca. The carboxyl content was measured using conductance titration (T50, Mettler Toledo) with 0.1 M NaOH. Isothermal titration calorimetry (ITC, ITC200, Malvern) was utilized to confirm the adsorption process and integrating ability between PION and ions. The concentration of COOH on the nanoparticles was diluted to 1.5 mM in cells and 4 mM CaCl2 and MgCl2 in the syringe with a titration injection of 2 μL per drop at a temperature less than 25 °C.

2.6. In vitro electrolyte concentration and osmotic pressure turbulence First, SBF solution was prepared consisting of 8.035 g of NaCl, 0.355 g of NaHCO3, 0.174 g of K2HPO4, 0.311 g of magnesium chloride hexahydrate, 0.072 g of anhydrous sodium sulfate and 6.118 g of HEPES. 1 M NaOH was used to adjust the pH value to 7.4. Then, the vacuum agitated filter was installed. The membrane diameter was 47 mm, and the molecular weight cut off was 100 kDa. In vitro osmotic pressure was measured using the following procedure: 450 μL of PION and PION-Ca with a concentration of 10 mg/mL were added to 1.3 mL SBF, separately. A 50 μL sample was placed on the osmometer to check the osmotic pressure at different times: 1 min, 3 min, 5 min, 1 h, 12 h, and 24 h. Three replicates were obtained at each time point and an analysis was performed. 2.7. In vivo ion balance analysis Rats were harvested from the inferior vena cava and several SD rats with a clean grade that were the same age, weighed 200 ± 20 g, were housed in a culture temperature 20 °C with a relative humidity of 45% and day and night cycle of 12 h. Prior to administration, the rats were anesthetized with 1% sodium pentobarbital at 900 μL/mouse and fixed on a wooden board after anesthesia. PION and PION-Ca were administered via a tail vein injection. At approximately 5 min after the injection, blood was collected from the abdominal vena cava using a vacuum blood vessel 3 times. The removed blood was transferred to a vacuum tube, rapidly centrifuged at 4 °C 3000 rpm for 10 min, and then the serum was separated. The serum was stored in a refrigerator at 4 °C for further analysis.

2.4. Cell viability assay and cellular internalization The cytotoxicity of PION was analyzed in L929 mouse fibroblast cell line at the concentrations of 12.5, 25, 50, 100, 200 and 400 μg/ml for 24, 48 and 72 h. In brief, L929 cells were seeded at 5 × 103 cells per well in 96-well plates and incubated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS. After 24, 48 and 72 h, 100 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and then all 96-well plates were incubated at 37 °C for 4 h. After removal of unreacted MTT, 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve participates in each well. The absorbance was measured at 490 and 630 nm via a Microplate Spectrophotometer (BioTek Eon, Vermont, USA). The L929 cells were seeded at round coverslips in a 24-well plate at

2.8. Influences of PION on heart functions evaluated by ultrasound in vivo Several SD rats were prepared, and they were all males weighing approximately 180–200 g. The selected anesthetic was 1% sodium pentobarbital, and each rat was injected with 1 ml for anesthesia. The rat and the fixation plate were placed on a bed beside the diagnostic apparatus, and the ultrasonic conductive gel was applied to the chest of the rat. Then the ultrasonic probe was placed at the heart position of the rat for imaging. B-mode ultrasound and CDI were performed by an expert sonographer (X.P.) using a color Doppler unit and a 9- to 12-MHz linear-array transducer (Siemens AG, Erlangen, Germany). First, a 3

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grayscale ultrasound was performed, which demonstrated anatomic details and sonographic characteristics. Next, a color doppler examination was conducted, which revealed the existence and changes in cardiac blood flow. Rats were examined in the supine position. An abundant quantity of gel was applied to cover the chest where the heart was located. The cardiac blood flow changes were also observed by via dynamic vector blood flow (V-Flow) (Resona 7, Mindray, China). After locating the position of the heart with B-mode ultrasound, the observation method was changed to V-flow mode. Rats that were administered saline, PION and PION-Ca were recorded for 10 min post injection.

one week, spontaneously beating cells could be observed. Cells were used for further experiments. The experimental current recording scheme was as follows. When the whole cells were sealed, it was switched to the current clamp mode. The cell membrane current was clamped at 0 pA, and spontaneous action potentials of the cardiomyocytes were continuously recorded. The experimental data were collected with an EPC-10 amplifier (HEKA). 2.12. Systematic toxicity evaluation of PION-Ca Mice were first administered with PION-Ca. After the animals were sacrificed, part of the heart, lung, liver, kidney and spleen were taken and fixed in a 4% paraformaldehyde solution. After 24 h, the fixative was removed. The fixed tissue was sampled, dehydrated, made transparent, embedded in paraffin, sectioned and subjected to HE staining and Prussian blue staining. Blood samples were centrifuged to obtain serum for biochemical analysis. An automated biochemical analyzer was applied for detection of several biochemical indicators, i.e., creatinine (Cr), alkaline phosphatase (ALP) and alanine aminotransferase (ALT). Furthermore, the metabolism of PION-Ca was investigated after injection 200 mg/kg in mice. Tissues were digested with aqua regia for 24 h and Fe concentration was measured using AAS.

2.9. In vivo electrocardiogram recording A number of SD rats were prepared under the same conditions as above. Prior to administration, rats were anesthetized with 1% sodium pentobarbital in an amount of 900 μL/mouse. After being given the anesthesia, they were fixed on a wooden board. PION was autoclaved prior to use, and 1 ml CaCl2 was added to 7.5 ml sterilized PION to form PION-Ca. Saline, PION and PION-Ca was administered by tail vein injection. Electrocardiogram recording was performed using the limb lead. Three electrodes on an ECG monitor were inserted into the subcutaneous tissues of the upper limb of the rats, and electrocardiography was performed before and after injecting the samples. Balb/c mice were prepared and fed at 20 °C with a relative humidity of 45% and a day and night cycle of 12 h. The mice were anesthetized with 1% sodium pentobarbital at 200 μL per mouse. Then, saline, PION, and PION-Ca were administered by tail vein injection. Electrocardiography recording was the same as above.

2.13. Injection safety study on beagle dogs The body weight of the animal was weighed before administration, and the dose for each animal was determined according to the body weight of the animal using a suitable disposable syringe and an intravenous infusion needle. The injection dose of PION and PION-Ca was 50 mg/kg with a Fe concentration of 10 mg/mL and injection volume of 5.58 mL. A single administration was used in this test and animals were weighed 1 day before the injection. Before administration, the telemetry system, parameters, implant and various physiological indexes were set. Blood pressure, ECG, respiration and other indicators were collected within 8 h after administration. At approximately 8 h after the drug was administered and after the data collection was completed, the telemetry system was turned off and the switching system time was recorded. The ECG indicators were QT interval (ms), heart rate (bpm), and PR interval (ms). Blood pressure indicators were systolic blood pressure (mmHg) and diastolic blood pressure (mmHg). The respiratory index was respiratory rate (BPM). The detection time points were as follows: 1 h before drug administration and 5 min ( ± 2 min), 10 min ( ± 3 min), 15 min ( ± 5 min), 20 min ( ± 5 min), 30 min ( ± 5 min), 45 min ( ± 5 min), 1 h ( ± 10 min), 2 h ( ± 15 min), 5 h ( ± 20 min), and 8 h ( ± 0.5 h) after administration.

2.10. In vitro recordings of CaV1.2, hERG and NaV1.5 ion channels by patch clamp For CaV1.2, the whole-cell patch clamp was used to record the L-type calcium channel current. The voltage stimulation protocol was as follows. When the whole cell was sealed, the cell membrane voltage was clamped at −60 mV. The clamping voltage was deducted from −60 mV to +10 mV for 0.3 s (specifically, the depolarization voltage reference pilot IV test). Data were collected every 20 s to observe the effect of the drug on the L-type calcium channel current peak. The experimental data were collected with an EPC-10 amplifier (HEKA). For HERG, a whole-cell patch clamp was used to record the whole-cell hERG-potassium. The voltage stimulation protocol was as follows. The cell membrane voltage was clamped at −80 mV when whole cell sealing was formed. The clamping voltage was diverted from −80 mV to +30 mV for 2.5 s and then quickly held at −50 mV for 4 s to excite the tail current of the hERG channel. Data were collected every 10 s to observe the effect of the drug on the hERG tail current. Experimental data were collected with an EPC-10 amplifier and stored in PatchMaster software. For NaV1.5, the whole-cell patch clamp was used to record the NaV1.5 sodium channel current. The voltage stimulation protocol was as follows. When the whole cell was sealed, the cell membrane voltage was clamped at −120 mV and the clamping voltage was maintained from −120 mV to −30 mV for 0.25 s. The data were collected repeatedly for 10 s to observe the effect of the drug on the peak current of the NaV1.5 sodium channel. Experimental data were acquired with an EPC-10 amplifier and stored with PatchMaster software.

2.14. In vitro and in vivo imaging efficacy evaluation T1 and T2 contrast effect of PION-Ca was evaluated in vivo and in vitro. Relaxation time experiments were recorded using an NMR analyzer Bruker mq 60 (60 MHz, 1.41 T) at 37 °C at different Fe concentration (0.2, 0.6, 1.0, 1.5, 2.0 mM). The slope was relevant relaxation rate. In vitro and in vivo MRI images of nanoparticles were obtained on a 3T GE Medical Systems (discovery MR750w) with a GEM Flex Coil 16-m. Ana-1 cell were used to confirm the optimum detection time of MRI in vivo. Cells were incubated on a coverslip with 1 mM PION-Ca for different times (0, 3.5, 6, 10, 15, 24 h) and then dye with Prussian. Photos were taken using Fluorescent Inverted microscope (Cossim). Cells were counted using Flowcytometry and Fe concentration was detected using AAS. Rabbits were used to image magnetic resonance angiography (MRA) and MRI at popliteal lymph node images. Animal tests were allowed by the animal care center and use committee of Shanghai Jiao Tong University. MRA were taken by using a 3D Fast Spoiled Gradient Echo (3D-FSPGR-TOF) sequence (TR = 5.4 ms, TE = 1.6 ms, filp angle = 30°, FOV = 10 cm2, slice thickness = 0.8 mm). Popliteal lymph node images were applied

2.11. Investigation of action potential changes on IPSC-CMs by patch clamp Stem cell-induced differentiation of cardiomyocytes was performed according to Cauliscell's standard procedure for cardiomyocyte processing. The brief description of the procedure follows. Stem cells were seeded in a 24-well plate with a density of 62,500/well under a 0.1% gelatin plate for 24 h and incubated in a 37 °C 5% CO2 cell culture incubator. The culture medium was changed every two days. After about 4

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with 3D Gradient Echo(3D-GRE)sequence(T2WI: TR = 8.96 ms, TE = 3.21 ms, filp angle = 30°, FOV = 100 mm, slice thickness = 2 mm). Pentobarbital sodium with a dose of 45 mg/kg was used in rabbits' anesthesia through ear vein injection. The dose of Gd-DTPA was 1 ml/kg. The dose of injected PION-Ca was 150 μmol Fe per kg and images were gained immediately after injection at abdominal aorta for MRA imaging and popliteal lymph node for MRI after different times of 13 h, 24 h, 45 h, 96 h.

PION, synthesized using a microwave-assisted polyol process was designed by our group [25,26]. The basic physicochemical properties of PION were characterized. The morphology of PION has been characterized by TEM and high resolution TEM (HRTEM). As shown in Fig. 1A, the synthesized PION particles are mono-dispersed on a copper grid without any agglomeration, indicating that PION has good dispersity in water. HRTEM in Fig. 1B reveals well-defined lattice planes and interplanar distances of 2.533 Å, which stand for (311) planes of Fe3O4 [29]. The statistical data in Fig. 1C indicate the average size of PION is 5.13 ± 1.0 nm with a narrow size distribution. TEM and HRTEM of PION-Ca have been shown in Figs. S1A and B. No agglomeration was found and crystal face distance was in accordance with PION, which means adding Ca ions would not influence Fe3O4 core. The average size calculated from (d311) using Debye-Scherrer's equation was 5.29 nm in both, which is in accordance with the TEM results. Furthermore, XRD pattern was applied to analyze the crystal structure of nanoparticles and this is presented in Fig. 1D for PION and Fig. S2A for PION-Ca. All diffraction peaks of the nanoparticles coincided well with magnetite from the JCPDS card (No.39–1346). The two sharp peaks in PION-Ca were the characteristic peaks of CaCl2 (Fig. S2A) [30]. Fig. 1E displayed FTIR spectra of PION (red) with blank PAA

2.15. Statistical analyses All data are the average ± SD of experiments repeated at least thrice. If imparity was statistically significant, a Student's t-test was used for verification. A p value of < 0.05 indicated significant differences between the groups. 3. Results and discussion 3.1. Characterization of PAA decorated iron oxide nanoparticles (PION) PAA decorated superparamagnetic iron oxide nanoparticles, named

Fig. 1. Characterizations and structural transformation of PION. TEM (A) and HRTEM (B) of PION. (C) Quantification of size distribution of PION calculated from HRTEM. (D) XRD patterns of PION. (E) FTIR spectrum of PION (red), PAA (blank) and DEG (blue). (F) Magnetization curves of PION at room temperature under ± 2T, inset is corresponding magnetization curves at low magnetic field. (G) Hydrodynamic size distribution before and after adding Ca2+. (H) Zeta potential of PION and PION-Ca. (I) Corresponding conformation of PION before and after adding Ca2+. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 5

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(black) and DEG (blue) as a control. For blank PAA, 1723.7 cm−1 is a characteristic peak, which represents C]O stretching of the carboxylic group [31]. However, the relative intensity of the 1723 cm−1 peak in PION almost vanished and two new bands at 1632.4 and 1567.1 cm−1 appeared, which were attributed to the asymmetric and symmetric stretching vibration of the COO− anion group [32,33]. This manifested that PAA was coated on the particle surface successfully. Peaks at 1126 cm−1 and 1059 cm−1 in the DEG spectrum were C-O-C and C-OH vibrations, respectively [34]. In PION, these two peaks disappeared, demonstrating PION was rinsed completely. The magnetic property of PION and PION-Ca were detected using VSM and the corresponding results are shown in Fig. 1F and Fig. S2B. The insert is the magnetization under a low magnetic field. The saturation magnetization of PION was calculated to be 59 emu/g and the curve had no remanence as well as coercivity, indicating its superparamagnetic property [35]. After adding Ca2+, the saturation magnetization decreased to 51 emu/g, this may be caused by CaCl2 that around nanoparticles surface that coating may decrease saturation magnetization [36]. The hydrodynamic diameter of PION and PION-Ca (pre-adding Ca2+ into PION, n (Ca): n (COOH) = 3: 8) were monitored, it was 40 nm for PION and 28 nm for PION-Ca in Fig. 1G. Ca2+ could be coordinated with COO− on PAA chains, which induced the length of PAA chain contractions [37,38]. The DLS of PION-Ca is smaller than PION [39,40]. Zeta potential of PION (−14.8 mV) increased after adding Ca2+ (−8.3 mV) (Fig. 1H), and the structural change of the PAA chains is shown in Fig. 1I. The

chemical structure of PION was brush-like with numerous COOH on the surface, after adding Ca2+, PAA chains will collapse as showed in Scheme 1 and Fig. 1I [41,42], which was speculated from the changes of size distribution and zeta potential (Fig. 1G and H). Paw swelling and MTT assays of PION are depicted in Figs. S3 and S4. With increasing concentrations of PION, the paw swelling volume was constant even at high dose (135 μmol/kg), which illustrates that injection of PION does not induce inflammation. L929 cell viability was above 85% even at high concentrations and a long period of incubation, which shows low toxicity to cells of PION. These results demonstrate the safety of PION in causing inflammation and toxicity in cells. Cellular uptake of PION-Ca was also taken as depicted in Fig. S5 by confocal microscopy. Results presented that the internalization of PION-Ca@5-AF by L929 cells was in a time dependent profile (Fig. S5A), which was confirmed by the quantification of fluorescence intensity of 5-AF (Fig. S5B). Meanwhile, the fluorescence intensity of DAPI showed no differences between each time scale (Fig. S5C). The stability is critical for both PION and PIONCa. The stability of PION was investigated by C. Miao et al., which PION was stable when subjected to PBS and SBF solution for 1 month and even had no obvious changes after storage for 1 year at room temperature [25]. Then, stability of PION-Ca was carried out by incubated with FBS (serum stability) and water (storage stability) under 4 and 37 °C [28]. No obvious aggregation of PION-Ca was observed until incubated for 240 h with FBS at 37 °C (Fig. S6A). When incubated with FBS at 4 °C, PION-Ca showed excellent stability at all time scales (Fig.

Fig. 2. Influences on ion balances of PION in vitro. Influences on Ca (A) and Mg (B) in simulated body fluid of PION and PION-Ca at different time points. (C) Osmotic pressure changes influenced by PION and PION-Ca. (D) and (E) ITC curves of 1.5 mM COOH in PION under pH 7.4 titrated by 4 mM Ca2+ and Mg2+. (F) ITC plots of 1.5 mM COOH in PION-Ca under pH 7.4 titrated by 4 mM Mg2+. 6

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S6A). Results from serum-induced aggregation assay validated that PION-Ca possessed out-standing stability in serum condition. The storage stability of PION-Ca was also investigated for 10 days. The average diameter of PION-Ca showed no obvious changes during storage for 10 days at 4 °C (Fig. S6B). However, the diameter changed significantly after 4 days of storage at 37 °C (Fig. S6B), which means that PION-Ca is more suitable storing at 4 °C. The representative size distribution of PION-Ca storage with water at 4 and 37 °C was presented at Figs. S6C and D, respectively.

may be much safer in vivo. 3.3. Revealing of cardiac dysfunction caused by interaction between physiological ion and PION The balance of electrolytes in our body is crucial for normal functioning of our cells and organs. Pathological changes occur in our body when the balance is broken [43,44]. In vivo normal concentration range of K+, Ca2+, Na+, Mg2+, and Cl− is 3.5–5.5 mmol/L, 2.25–2.75 mmol/ L, 136–145 mmol/L, 0.6–1.1 mmol/L, and 98–106 mmol/L, respectively. Hence, the influence on ion concentration was also investigated in vivo. PION/PION-Ca was injected intravenously with a rate of 2 ml/s into SD rats, and blood samples were extracted after 5 min. The process is depicted in Fig. S9. Serum was obtained by centrifugation and a magnetic separation column. Corresponding results are exhibited in Fig. 3(A and B). The concentration of Ca2+ and Mg2+ after injecting PION in vivo was decreased, indicating that PION absorbed serum Ca2+ and Mg2+. As Mg2+ is an essential kinase factor for the Na+-K+-ATP enzyme, if Mg2+ is lacking, this enzyme would lose activity, which results in K+ being released from the cell that induce K+ increase (Fig. S10A). As the total cation concentration decreased, Cl− also decreased due to an electrolyte balance in vivo (Fig. S10C). Except for Na+, all the ions were out of the normal range in serum, demonstrating PION could break the ion balance, which may further influence body safety (Fig. S10B) [45–47]. The increase in the concentration of Ca2+ was due to some of the absorbed ions being released to the blood to reach a Ca2+ balance (Fig. 3A). Although K+ (Fig. S10A), Mg2+ (Fig. 3B) and Ca2+ (Fig. 3A) concentrations were increased in the serum, there was no obvious side effect observed in the rats. All in all, PION-Ca had a lower effect in vivo compared to PION. Fig. 3C shows the acute toxicity of PION and PION-Ca at different doses from 50 to 350 mg/kg in mice observed for 2 days. The maximum tolerated dose of PION-Ca was as high as 250 mg/kg, which was 100-fold over the MRI efficient dose, and it is 2.5 times higher than PION (100 mg/kg). These results demonstrated that coordination with Ca2+ in advance could enhance the particle safety greatly. Within some death incidents of the mice (pink frothy sputum), a sign of serious heart failure appeared with PION, as shown in Fig. S11. As the electrolyte balance could affect cardiomyocytes, the mechanism of PION on cardiac toxicity is described below [46–48]. To further and directly observe the effects of PION on animal heart function [49], SD rats were selected as model animals. Under B-mode ultrasound monitoring, the heart rate of rats was stabilized at 330–340 beats/min before injecting PION samples (Fig. 3D). After injecting PION, the heart beat weakened and then dropped rapidly (Fig. 3F), while a high reflection shadow soon appeared in the ventricle in the heart of the rats. It is presumed that the shadow in the heart revealed a blood flow jam, and the significant change in the heart rate resulted in serious heart failure (Fig. 3D, F) [46,50,51]. During B-mode ultrasound monitoring, after PION-Ca was injected (Fig. 3E, G), the heart rate of rats stabilized at approximately 340 beats/min. Blood flow was quantified by color doppler and other indicators did not present a significant impact. At the same time, the ventricle in the heart was clear with no high intensity reflections appearing and blood flow filled the heart without abnormalities (Fig. 3E). After saline was injected, the blood flow and blood flow direction of the rats were not significantly affected (Figs. S12A and B), which indicated that injection of 4 ml of normal saline did not affect the function of the rats' hearts. Pulsatility index (PI) and resistance index (RI) were also measured and are shown in Figs. S13 and S14. Both PI and RI increased significantly after being treated with PION, indicating that the normal blood flow conditions were disturbed [52] and that the administration of PION could induce serious heart failure [46]. PI and RI showed no significant changes after injection with PION-Ca, confirming the viability of evading the toxicity of PION by pre-chelation Ca2+ (Figs. S13 and S14) [52]. Later, Blood flow quantified by color doppler also showed no significant variation at 1, 24

3.2. Interaction between PION and multiple ion in vitro As PAA is a polyelectrolyte and has the characterization of a counter-ion confinement effect, it can absorb ions in bulk solution. The absorption situation was investigated in vitro at different times after adding PION and PION-Ca samples (Fig. S7), where PION-Ca was prepared by adding CaCl2 (n (Ca): n (COOH) = 3: 8) to PION. As depicted in Fig. S8A, K+ decreased over 40% after adding both samples and PIONCa recovered tardily, while there was no obvious change in PION. Due to the abundant COOH on particle surfaces, K+ was coordinated with COOH. The influence on Na+ was similar to K+, while Na+ declined by approximately 20%, this is because some Na+ existed in the PION and PION-Ca samples (Fig. S8B). PION could absorb both Ca2+ and Mg2+ in simulated body fluid (SBF) with a decreased percentage of approximately 68%. However, when supplemented with Ca2+ before injection, some of the absorbed Ca2+ released to the bulk solution, which induced the Ca2+ increase due to the ion balance (Fig. 2A). Only approximately 6% of Mg2+ decreased after adding PION-Ca, which demonstrated PION had little effect on Mg2+ concentration after coordination with Ca2+ (Fig. 2B). As all the cations were decreased, some of the absorbed ions also integrated with Cl− (Fig. S8C). Hence, the concentration of Cl− decreased approximately 40% for PION. Fig. 2C shows the influence of PION and PION-Ca on the osmotic pressure. For each liquid, the osmotic pressure was constant at different time points except for a little decrease in PION + SBF due to the absorption process. PION + SBF soon became constant by 3 min, which meant ion balance was a quick process. Furthermore, PION appeared to have the lowest osmotic pressure and it was approximately 81% lower than saline and SBF, while PION-Ca was only 33% lower than saline and SBF. When SBF was used as a solvent, PION + SBF rose to 74% saline and PION-Ca + SBF increased to normal value (Fig. 2C). According to the osmotic pressure variation, cation ion binding tendency of PION was explicable by the presence of Donnan effect [40,42]. To further investigate the absorption effect between PION and ions, isothermal titration calorimetry (ITC) was used. From the thermal effect, ITC could give the thermodynamic parameters of binding stoichiometry (n), the binding constant (K), changes of entropy (ΔS) and enthalpy (ΔH). Peaks in the thermogram corresponded to the endothermic reaction heat effect, while the corresponding Wiseman diagram was integrated from the peak area of the thermogram and were fitted by nonlinear least-squares. As shown in Fig. 2D-F, strong interactions of PION with Ca2+ and Mg2+ were clearly observed from the sharp endothermic peaks and the binding site n was 0.110 and 0.099 for Ca2+ and Mg2+, respectively. This indicated that there were approximately 10 COO− groups combined with 1 ion under pH 7.4. The free energy change could be calculated using equation ΔG = ΔH – TΔS, where T is the cell temperature, and the results were −6.2 kcal/mol for Ca2+ and −6.3 kcal/mol for Mg2+, this means it was a spontaneous interaction between PION and ions. The absorption ability was almost the same between Ca2+ and Mg2+. As depicted in Fig. 2F, no obvious signal change was observed, which means PION-Ca had little effect on Mg2+ absorption, which was in accordance with the in vitro Mg2+ concentration after adding PION-Ca in SBF. All these results suggested that PION could combine with ions and rapidly reached to ion balance. PION-Ca had little effect on ion concentration, especially for Mg2+, and it was much closer to a normal osmotic pressure, indicating PION-Ca 7

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Fig. 3. Impacts on blood ion concentrations and heart function detected by in vivo acoustic detections of PION and PION-Ca. Influences on Ca2+ (A) and Mg2+ (B) ions in the serum of PION and PION-Ca at different time points. (C) Acute toxicity of PION in rats before and after adding Ca2+ at a dose range from 50 to 350 mg/kg (D, E) color doppler that measured the blood flow changes in hearts of rats. Red dotted and white dotted lines indicated the heart morphology and color doppler measurement area, respectively. (F) and (G) the quantification results of heart rates after administered with PION and PION-Ca. (H) Ultrasound scanning of rats' heart blood flow through V-flow after being injected with saline, PION and PION-Ca. Data are presented as the means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 8

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and 48 h post PION-Ca injection, which further certificated the safer application of PION-Ca over PION at longer time scale (Fig. S15A). The HR, PI and RI indexes also in accordance with ultrasound observation (Fig. S15B, C and D). Moreover, dynamic vector blood flow (V-Flow) was applied to monitor blood flow changes [53,54]. As shown in Fig. 3H, no changes were showed after injection of saline (Movie S1 and S2). However, the V-flow (blue arrow) changes dramatically when PION was administered. The dynamic blood flows nearly disappeared after 5 min, which was regarded as a sign of serious heart failure (Movie S3 and S4). Hence, serious heart failure is the main reason for the animals' deaths. After administration of PION-Ca, the V-flow showed no obvious changes in heart blood flow (Fig. 3H, Movie S5 and S6). Comparing between PION and PION-Ca, pre-chelation Ca2+ sealed the binding point of PAA and prevented PION from chelating with other bivalent ions, thus the blood ion balance was stabilized and the

electrophysiological function of the heart was protected. Further, the dynamic blood flow also did no change significantly at 1, 24 and 48 h after PION-Ca administration (Fig. S15E, Movie S7, S8 and S9). These results suggested that the serious heart failure caused by an ion imbalance mainly accounted for the animals’ deaths [55]. However, preadding Ca2+ effectively inhibited heart failure caused by PION, which indicated a feasible solution for reducing lethal toxicity in vivo. Supplementary data related to this article can be found at https:// doi.org/10.1016/j.biomaterials.2019.119442 3.4. Cardiac dysfunction induced by PION presented in electrocardiography aspect Based on the above experimental results, we preliminarily speculated that the abundant COOH groups on the surface of PION had a

Fig. 4. Electrophysiological variations of the heart caused by PION and PION-Ca (A) Impacts on electrocardiograms of mice at pre-injection and after-injection of saline, PION and PION-Ca. (B) Process of IPSC induced cardiomyocytes regarding action potential related parameters. (C) Action potential repolarization of 90% of the membrane potential level and heart rate of PION. (D) The action potential repolarization of 90% of the membrane potential level and heart rate of PION-Ca. Data are presented as the means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). 9

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strong affinity to cations in vivo, especially for Ca2+ and Mg2+ ions. Therefore, electrocardiography (ECG) was applied to reflect the effect of PION on cardiac electrophysiology, as shown in Fig. 3A, which can further determine whether cardiac function was affected by breaking of the ion balance [48,56]. Serious typical atrioventricular block (AVB) patterns are shown in Fig. 4A (red arrows), which shows that the strong influence of PION on the ion balance can induce deadly heart failure caused by strong AVB [57]. A preliminary conclusion could be drawn according to the ECG experiment in that the high affinity to bivalent ions of PION, which was caused by the high amount of surface polyacrylic acid decoration, strongly interfered with the ion balance in the blood and led to serious arrhythmia and heart failure. To further support this conclusion, replicated experiments were carried out on SD rats (Fig. S16). There was no significant change in the electrocardiograms after being injected with saline in rats, indicating that saline did not cause changes in the ECG results. However, when PION was administered, the QRS complex, representing the ventricular depolarization process, changed dramatically, which suggested that the inward T-type Ca2+ ion current flow was affected. Ventricular tachycardia (red arrows) and typical patterns of an atrioventricular block (AVB) (green arrows) are shown in Fig. S16. T wave flattening (blue arrows) also indicated the Ca2+ concentration may be affected [58]. On the other hand, when injected with PION-Ca, no significant changes in the ECG results were found, demonstrating that PION-Ca had negligible impact on the blood ion concentration and heart electrophysiology [58]. All of these phenomena strongly suggested that PION had influence on ion concentration can lead to serious AVB, which caused the deaths of experimental animals (Fig. S16). In view of the fact that PION may cause severe arrhythmia and heart failure, an investigation was carried out on the impact of PION on the action potentials of cardiomyocytes that were differentiated from human induced pluripotent stem cells (IPSC) [57,59,60]. Scheme of the process is shown in Fig. 4B with corresponding action potential repolarization of 90% and heart rate shown in Fig. 4C and D. At low

concentrations (1 μM), PION showed no significant effect on the action potential durations of APD30, APD50 and APD90 of cardiomyocytes (Fig. 4C, Fig. S17). The action potential durations (APD 30, APD 50 and APD 90) of cardiomyocytes under higher concentrations of PION (3 μM, 10 μM and 30 μM) were significantly shortened, suggesting that the strong adsorption capacity of PION on bivalent ions had a significant impact on the electrophysiological activity of cardiomyocytes [61,62]. Furthermore, due to the shortening of APD, PION concentration-dependent acceleration of the spontaneous pulsation (HR) (Fig. 4C) of cardiomyocytes was observed [63], and the increase of HR could reflect the increase of cardiomyocytes excitability due to the decrease of Ca2+ [62]. The concentration of PION on cardiomyocytes had no significant impact on the action potential 0 phase depolarization amplitude (APA) and resting membrane potential (RMP) (Figs. S18A and B). In addition, no obvious effect of APD was shown in the PION-Ca group (Fig. 4D, Fig. S19). Moreover, PION-Ca also had no significant effect on the action potential 0 phase depolarization amplitude (APA) and resting membrane potential (RMP) of IPSC-induced cardiomyocytes at all concentrations (Figs. S20A and B). All the results above illustrated that the interference of PION on the ion balance greatly disturbs the function of cardiomyocytes at a cellular level and then caused arrhythmia and heart failure at a macro level. Fortunately, by pre-chelation Ca2+, the safety of PION is greatly improved on an electrophysiological level. 3.5. Variations of typical ion channels caused by PION To further qualify the influence of PION on a cellular level, evaluations of a typical cardiac ion channel were carried out. The evaluation on a typical ion channel is also a fundamental step for the development of new drugs. PION exhibited a greater inhibition effect on the CaV1.2 calcium channel than PION-Ca and the inhibition also showed a concentration dependent trend. Therefore, the effect of PION on CaV1.2 calcium ions expanded the possibility of causing an arrhythmia (Fig. 5A and B) [61,64,65]. The effects of PION and PION-Ca

Fig. 5. Electrophysiological influences on vital ion channels caused by PIOH. (A) Current change of CaV1.2 induced by PION and PION-Ca and quantification results of the inhibition rate is shown in (B). (C) Current change of hERG induced by PION and PION-Ca and quantification results of the inhibition rate is shown in (D). (E) Current change of NaV1.5 induced by PION and PION-Ca and quantification results of the inhibition rate is shown in (F). Data are presented as the means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). 10

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Fig. 6. In vivo safety in mice and effects of single intravenous injection of PION and PION-Ca on cardiovascular and respiratory function in beagle dogs. (A, B, C) Serum parameters of ALP, ALT, and Cr in mice. (D and E) Prussian blue staining and HE staining of the main organs (100 × ). Effects on systolic blood pressure and diastolic blood pressure (F). Impacts on the PR interval and QT interval (G). Impacts on the breath rate and heart rate (H). Data are presented as the means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

on hERG ion channel are shown in Fig. 5C and D, which showed no significant impact on potassium ion channel current in both samples, demonstrating that PION and PION-Ca have no significant inhibitory effect on hERG channels and will not cause potassium channel-

dependent arrhythmias [66]. As presented in Fig. 5E and F, no obvious impacts on NaV1.5 ion channels were found, illustrating that no sodium channel-dependent arrhythmia was found. In summary, as PION has a significant adsorption effect on Ca2+, the cross-membrane current 11

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mediated by CaV1.2 ion channels are significantly inhibited, which is a possible reason that the arrhythmia leading to animal death. Accordingly, the results so far strongly suggest that cardiac toxicity caused by PION is mainly determined by the inhibitory effect on the CaV1.2 calcium ion channel, for the open/close of this L-type calcium channels are controllable by blood calcium levels. All the ion channel models (hERG, NaV1.5 and CaV1.2) were validated by the positive controls Cisapride, TTX and Nifidipine, respectively (Fig. S21).

was present 3 days post-administration of PION-Ca. These results were consistent with previous studies that showed the injection of PION did not result in detectable damage in the main organs (Fig. 6E). The metabolism of PION-Ca was investigated in Fig. S22. After 24 h, the concentration of Fe in tissues reached the highest 4.4, 34.8, 32.2, 12.8, 3.8 mmol/kg for heart, liver, spleen, lung, kidney, respectively. At day 21, more than 2/3 Fe was metabolized in liver and spleen. Fe in heart, lung and kidney were fully metabolized, the metabolism rate of PIONCa was suitable, which realizing a longer imaging period while further guaranteeing a safe in vivo application [67]. Bolus injection of PION-Ca is crucial and required for further safety testing in other animal models. Beagle dogs were chosen to be the model animal for this section of investigation. The clinical observation was made after injection, and the animals were subjected to general toxicity observation before and on the day of administration. During the test, animals were intravenously injected with a 50 mg/kg dose of PION and PION-Ca. After being injected with PION, the systolic blood pressure and diastolic blood pressure decreased dramatically and lasted for over 1 h (Fig. 6F). Although PION-Ca also induced a slight transient decline in blood pressure, the parameters of systolic and diastolic blood

3.6. Systematic biocompatibility and injection safety evaluation in vivo Serum parameters (ALT, ALP, Cr) were also used to evaluate the systematic toxicity of PION-Ca in mice. As shown in Fig. 6A, B, and C, no significant differences occurred by ALT, ALP and Cr between PBS and PION-Ca groups after 3 days of administration, suggesting that PION-Ca would not affect the liver and kidney functions of mice. Prussian blue staining sections were also observed under a microscope, as shown in Fig. 6D. There was an obvious blue area in liver and spleen, which indicated that PION-Ca was mainly distributed in liver and spleen. Moreover, HE staining confirmed that no visible tissue damage

Fig. 7. The application of PIOH-Ca in magnetic resonance angiography (MRA) and lymph nodes imaging. (A) MRA imaging of Gd-DTPA and PION-Ca. (B) MRI efficiency of PION-Ca in Lymph nodes visualizing before and after 24 h. The corresponding signal decreasing ratio (C) and signal change ratio (D) data collected from T2-weighted images. (n = 5). 12

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pressures soon recovered to a normal status after 5 min (Fig. 6F). The PR and QT interval of beagle dogs injected with PION were lengthened significantly, which could be regarded as signals of a minor atrioventricular block (Fig. 6G). The PR and QT intervals both recovered to a normal status after 1 h, while PION-Ca showed no significant influences on both PR and QT intervals. The breath rate of animals injected with PION showed dramatic augmentation, suggesting that heart failure had taken place. However, PION-Ca induced no changes in the breath rate of the beagles (Fig. 6H). Significant declination of heart rate was observed after being injected with PION, confirming the ability of PION to induce heart failure, while PION-Ca exhibited no impact on the heart rate. The series of results further strengthened the verdicts made previously in that PION is able to cause deadly heart failure and arrhythmia (atrioventricular block).

decreased of approximately 90%. Fig. 7C showed the signal decreased ratio of lymph node after injection PION-Ca at different times and reached to 90% at 13 h. The signal change also decreased dramatically, that is, it changed over 5-fold (Fig. 7D) before and after injection PIONCa for 24 h. These results exhibited the scan time point after injection was 24 h which in accordance with in vitro cell results. All these outcomes greatly stated the potential application of PION-Ca used as T1/ T2 contrast agent. 4. Conclusion In conclusion, there were several findings from the series of experiments in this study. First, due to the high intensity of COOH at the surface, PION has strong affinity to divalent ions, which influences the ion balance both in vitro and in vivo. The divalent ion binding tendency of PION is explicable by the principle of the Donnan effect and it is preventable by pre-chelation Ca2+ to PION (PION-Ca). Second, the maximum tolerated dose of PION-Ca is higher than 250 mg/kg, which is 100-fold higher than the MRI efficient dose and 2.5 times higher than PION (100 mg/kg). Finally, PION-Ca stabilized the blood ion balance and protected the electrophysiological function of the heart by sealing the binding points of Ca2+ and Mg2+. This was demonstrated by the Bmode and color doppler ultrasound monitoring and dynamic vector flow, ECG and patch clamp trials. To conclude, using PION or other PAA modified nanomaterials directly in vivo is risky due to the potential cardiac toxicity caused by ion balance intervention. The suitable and feasible option to prevent cardiac toxicity caused by such materials is pre-chelating biocompatible bivalent ions before injection. The experimental results suggest new ideas to avoid lethal toxicity and they could broaden further application of polyacrylic acid or COOH decorated nanoparticles in vivo.

3.7. MRI efficacy of PION-Ca in vitro and in vivo In vitro relaxation rate and MRI efficacy of different Fe concentration of PION and PION-Ca were measured using NMR recorder and GE MRI machine. As exhibited in Figs. S23A and B, the relaxation rate increased a little after adding Ca2+, which may cause by the collapse of PAA chains that make water molecule closer to iron surface. While the difference in MRI signal at different concentration of Fe between PION and PION-Ca in Figs. S23C and D was not obvious. In order to obtain better MRI efficacy, cellular uptake profile of PION-Ca in Ana-1 cells were undertaken (Fig. S24). From Fig. S24 A to F, cell Prussian blue staining showed that with increased incubation time blue area in cells increased and almost reached saturation after 15 h. Figs. S24G and H displayed relevant quantify results at different incubation time point. The total Fe concentration was increased as cells were proliferation during this time while Fe concentration per 10000 cells at 15 h and 24 h were close to each other, which meant the phagocytosis was almost saturated. Hence, the optimized MRI scanning time scale in vivo maybe 15–24 h. MRA effect was evaluated using PION-Ca and Gd-DTPA. For the first 30 s (first pass imaging), aorta all clearly seen in both samples (arrowhead), portal vein was distinctive after 30 s for PION-Ca and 3 min for Gd-DTPA (curve arrow) (Fig. 7A). The signal intensity and area decreased sharply in Gd-DTPA group while no obvious change in PION-Ca sample under the time scale of 30 min. These indicating the potential application of PION-Ca in MRA contrast agent (Fig. 7A), which presenting much more advantages over the existed Gd-DTPA. T2weighted MRI images of rabbit popliteal lymph nodes were obtained after injection of PION-Ca for 24 h with a dosage of 150 μmol Fe per kg. As shown in Fig. 7B, in comparison to pre-contrast images, the lymph node signal intensity was obviously decreased and the signal percentage

Conflicts of interest There are no conflicts of interest to declare. Acknowledgement This research was supported by the National Natural Science Foundation of China (No. 81972886, No. 81773272, No. 81572999 No. 81771839 and No. 81874479), the State Key Laboratory of Oncogenes and Related Genes (No. 91-17-20), Medical-Engineering Joint Funds from Shanghai Jiao Tong University (No. YG2017QN43), Shanghai Municipal Commission of Health and Family Planning (No. 20174Y0123).

Appendix PION PION-Ca MTD ITC PAA PI RI ECG AVB IPSC CMs IPSC-CMs APD APA RMP HR

Polyacrylic acid coated Fe3O4 nanoparticles Polyacrylic acid coated Fe3O4 nanoparticles with Calcium ions Maximum tolerated dose Isothermal titration calorimetry Polyacrylic acid Pulsatility index Resistance index Electrocardiogram Atrioventricular block Human induced pluripotent stem cells Cardiomyocytes Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Action potential duration Action potential 0 phase depolarization amplitude Resting membrane potential Heart rate

13

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The human Ether-à-go-go-Related Gene encoded potassium ion channel The voltage-gated sodium channel Tetrodotoxin Creatinine Alkaline phosphatase Alanine aminotransferase

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biomaterials.2019.119442.

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