Materials and Reagents
Newborn (6 g, 24 h) and adult male Sprague-Dawley rats (250–280 g, 8 w), as well as BALB/c-nu mice (15 g, 4 w) were used in this study. All animal experiments were approved by Institutional Animal Care and Use Committee of Department of Laboratory Animal Science of Fudan University (Grant No. 20160780A176), and confirmed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85 − 23, revised 1996). DATS, 3-aminopropyltriethoxysilane (APTES), LF, N-succinimidyl-S-acetylthioacetate (SATA), and Mal-PEG-NHS were obtained from Sigma-Aldrich (St Louis, MO). Other reagents were of analytical grade and used as purchased. All the solutions were prepared by Milli-Q water and deaerated with high-purity nitrogen.
Preparation of MIONs
The synthesis of MIONs followed our previously described protocol with minor modifications . Briefly, styrene (9 mL), methacrylic acid (1 mL), and deionized water (70 mL) were mixed and dispersed. The suspension was then stirred and heated to 75 °C for 30 min. Then 10 mL of potassium peroxodisulfate (10 mg/mL) was gradually added and react for 24 h at 75 °C. The obtained polystyrene nanoparticles were mixed with 30 mL of ethylene glycol and 170 mL of deionized water. After that, 450 mg of ferrous chloride, 110 mg of potassium nitrate, and 2 g of methenamine were sequentially added and heated to 80 °C under nitrogen protection. After cooling and centrifuging, collected nanoparticles were washed with distilled water to remove residual reagents, and gradually heated to 500 °C for 3 h. After cooling to room temperature, MIONs were obtained.
The surface modifications of MIONs consist of conjugating Mal-PEG-NHS with MION and conjugating LF with Mal-PEG-MION (Supplementary Figure. 1). Firstly, 5 mL of APTES was hydrolyzed under catalyzation of HCl (pH 4.0) to form silane polymer with reactive saline bond, then 30 mL of deionized (DI) water and 10 mg of MIONs were added. The mixture was stirred for 4 h at 65 °C under nitrogen protection, and was eluted with ethanol and DI water. Then 20 mg of Mal-PEG-NHS (Mw: 5000) was added to the silane coated MIONs, and the mixture was allowed to react for 5 h at room temperature after 30 min nitrogen bubbling, following dialysis against DI water.
LF was activated before conjugated to Mal-PEG-MION according to previous method . Briefly, LF and SATA were incubated in Hepes buffered saline (pH 7.0) at 1: 8 molar ratios for 1 h at room temperature under constant shaking. Free SATA was removed by centrifugal ultrafiltration with the help of Millipore UFC801096 tubes (12000 rpm, 30 min at 4 °C). SATA modified LF was reacted with 0.1 M hydroxylamine for 45 min at room temperature before coupling to maleimide then was incubated with Mal-PEG-MION for 2 h at room temperature. After incubation, MION-PEG-LF went through centrifugal ultrafiltration with a 30Kd cut-off tube to dislodge uncombined LF and other impurities. After that, MION-PEG-LF solutions underwent FT-IR spectroscopy assay to confirm the conjugation of Mal-PEG-NHS and LF to MIONs (Perkin Elmer Frontier FT-IR, spectra recorded from wavenumber 400–4000 cm− 1) (detailed in Supporting Information). The average amounts of LF conjugated to MION was estimated by enzyme linked immunosorbent assay (ELISA) kit method.
DATS was loaded into MION-PEG-LF based on the previous reported protocols [8, 10]. In brief, 1 mg of MION-PEG-LF and 1 mg of DATS were sequentially mixed in 5 mL of distilled water, followed by stirring for 9 h. Resident DATS were removed from the surface of MIONs by being washed with distilled water. Parts of cleaned [email protected] were freeze-dried under vacuum and weighed to count the loading efficiency of DATS (mass of drug loaded in nanoparticles/ mass of drug loaded nanoparticles × 100%). Other parts of [email protected] were reserved in saline solutions at room temperature ready for use.
The structure of [email protected] was analyzed by high-resolution transmission electron microscopy (HRTEM) images which were recorded by a JEM-2100F (JEOL, Japan) and a Tecnai T20 (FEI, USA) transmission electron microscope (TEM). The diameters of samples were acquired by averaging size of 50 nanoparticles in TEM images. Dynamic light scattering (DLS) Autosizer 4700 (Malvern, MA, UK) was used to measure the size distribution. The conjugations of Mal-PEG-NHS/LF to MIONs were confirmed by FT-IR method (Supporting Information). The samples were tested at 25 °C with concentration of 5 µg/mL.
Cytotoxicity assay of [email protected] was assessed using primary neonatal cardiomyocytes and neurons according to the previously described method [10, 26]. The culture of cardiomyocytes and neurons were described in Supporting Information. The [email protected] was diluted with culture medium to gain a concentration range from 0 to 100 µg/mL, and was added to 96-well flat-bottomed tissue-culture plate with cell seeded. After incubation for 24 h, medium was removed, and cells were washed with PBS, then the medium was replaced with cell counting kit-8 (CCK-8) solution (Dojindo Laboratories, Kumamoto, Japan). The absorbance of individual wells was measured at 450 nm by a microplate reader (Molecular Devices, FlexStation 3, CA, USA). The results were expressed as the mean percentage of cell viability relative to control.
In vitro Cellular Uptake
Fluorescent dye DiR was loaded to the MION-PEG-LF frameworks. The protocols of loading and the identification of [email protected] were described in the Supporting Information. Neonatal rat cardiomyocytes and neurons were treated with the [email protected] (50 µg/mL) in 24-well plates for 4 h. After washed with PBS for thrice, the cells were fixed by 4% paraformaldehyde for 15 min, penetrated by 0.1% triton-X 100 for 20 min, stained by cTnT (cardiomyocytes) and MAP-2 (neuron) for 8 h and 4', 6-diamidino-2-phenylindole (DAPI) for 15 min. Then the [email protected] (λex = 750 nm) inside cells were visualized using a fluoresce microscope (Olympus IX-71, Japan).
The in vitro release course of [email protected] was assessed by real time H2S-selective microelectrode assay: 5 µg/mL, 10 µg/mL, and 20 µg/mL of [email protected] was separately added to a glass chamber (World Precision Instruments, WPI, USA) containing PBS (100 mM, pH 7.4, 4 mL) with 2 mM of GSH at 37 °C. Then H2S formation was detected using ISO-H2S-2 sensor attached to an Apollo 1100 Free Radical Analyser (WPI, FL, USA). H2S release was real-time displayed by picoamps (PA) current curve for 120 min.
Protection Effects of [email protected] from Hypoxia Induced Damage in Rat Cardiomyocytes and Neurons
[email protected] (1 ~ 10 µg/mL) + GSH (2 mM) and saline of same volume (Control) were separately added to medium of cultured cardiomyocytes or neurons (n = 3). After incubation for 4 h, medium was removed and replaced with DMEM/F-12 without glucose and serum. Then cardiomyocytes/neurons were exposed to hypoxia (94% N2, 5% CO2, 1% O2) for 4 h in a CO2 incubator (Forma SERIES II WATER JACKET, Thermo Scientific, MA, USA), followed by reoxygenation (5% CO2) for 1 h. After which, the cell viability was evaluated by CCK-8 assay and compared with the Control.
After the hypoxia/reoxygenation procedure, lactate dehydrogenase (LDH) activities of each group were also measured to evaluate cytotoxicity using an assay kit (JianCheng, Nanjing, China) according to the manufacturer’s instructions. The absorbance was determined by a micro plate reader at 440 nm. The levels of cell apoptosis were also determined by flow cytometry by previous method .
Like almost all nanoparticles, intravenously administered MIONs are eventually cleared by mononuclear phagocytic system such as liver . To investigate the in vivo metabolism and biodegradation of [email protected], BALB/c-nu mice (15 g, 4 w, n = 6) were injected via the tail vein with 10 mg/kg of the [email protected] nanoparticles. Six mice were injected with same volume of saline and underwent same procedures as control. In vivo MRI scan was performed on 3-T MRI scanner (Discovery MRI 750, GE Medical Systems, Milwaukee, WI, USA) with an animal 8-channel phased-array coil with the following parameters: FSE T2 Fat Suppress, slice thickness: 1.0 mm, spacing: 0.1 mm, TR: 4167.0 ms, TE: 68.0 ms, refocus flip angle: 142°, echo train length: 24, and bandwidth: 31.25 kHz. T2-weighted MRI was used to detect the aggregation of nanoparticles in mouse livers by evaluating the calculated signal intensity. The signal intensities of nanoparticles at 6 h, 12 h, 24 h, 2 d, 4 d, 6 d, 8 d, 10 d, 12 d, and 14 d after injection were obtained in a region of interest (liver region) placed at the fixed site on matched slices. Signal to noise ratio (SNR) were calculated to compare the relative signal intensity.
Sprague-Dawley rats were injected with [email protected] (10 mg/kg) through tail vein (n = 6). Rats were euthanized at 24 h, 7 d, 14 d, and 30 d after injection, and brain, heart, liver, spleen, lung and kidney organs were harvested and fixed in 4% paraformaldehyde for 24 h at room temperature then embedded in paraffin and cut into 7 µm-thick slices. The sections were stained with H&E and imaged under a light microscope at × 40 magnification. Six high-power fields were randomly selected in each photograph. Hematological and serological examinations at 24 h, 7 d, and 30 d after injections of [email protected] (10 mg/kg) or same volume of saline (Control) were also evaluated (Supporting Information).
Effect of [email protected] on heart rates and blood pressure
Sprague-Dawley rats (n = 6) were anesthetized with medetomidine hydrochloride (Domitor, 250 µg/kg, IP.) and ketamine hydrochloride (Ketamine, 50 mg/kg, IP.). Carotid arteries were cannulated and connected to Model SMUP-E4 Bioelectric Signals Processing System (MFLab301) to display rat real time heart rates and blood pressure, which were recorded every 1 hour. [email protected] (10 mg/kg) or same volume of saline (Sham) was administrated by tail vein injection. Then change of blood pressure and heart rates was evaluated.
Heart/Brain Targeting Assessment of [email protected]
DiR was loaded to the MION-PEG-LF and MION frameworks. The protocols of loading and the identification of [email protected] and [email protected] were described in the Supporting Information. Immunofluorescence method was applied to study the in vivo targeting ability of [email protected] to brain and heart. Sprague-Dawley rats were injected with either [email protected] (10 mg/kg) or [email protected] (10 mg/kg) by tail vein. At 24 h after injection, rats were anaesthetized and sacrificed to obtain the brain and heart tissues. The brain and heart tissues were dewaxed and hydrated in dimethylbenzene and a graded series of alcohol, frozen and embedded in Tissue-tek O.C.T., and sliced (10 µm). Cardiomyocytes cytoplasm were stained with wheat germ agglutinin (WGA, 1: 100), neuron cytomembranes were stained with neurofilament protein NF200 (1: 100) and goat anti-mouse IgG (1: 200), and DAPI was used for labeling of nuclei. The slides were then mounted with an antifade medium and observed with a DMI 4000 fluorescence microscope (Leica Camera Co., Wetzlar, Germany) (detailed in Supporting Information).
Sprague-Dawley rats were anesthetized with medetomidine hydrochloride (250 µg/kg, I.P.) and ketamine hydrochloride (50 mg/kg, I.P.) (n = 6). Carotid arteries were cannulated for blood withdrawal. 5 mg/kg, 10 mg/kg or 20 mg/kg of [email protected] were separately administrated by tail vein injection. Blood (0.1 mL) was withdrawn at time intervals (0 to 12 h) after administration. The blood collected was anticoagulated with heparin sodium (50 U/mL) and centrifuged (3000 rpm, 15 min) to obtain plasma. As to the H2S concentration of [email protected] in plasma cannot be assessed by real time H2S-selective microelectrode assay, therefore it was measured by HPLC method by an Agilent Technologies HPLC (1260 infinity, CA, USA) with fluorescence detection (λex: 390 nm and λem: 475 nm) and an Eclipse XDB-C18 column (150 ⋅ 4.6 mm, 5 µm) within 24 h, which was previously described [7, 29].
Repeat the experiments, and Sprague-Dawley rats (n = 6) administrated by [email protected] (10 mg/kg) or same volume of saline (Vehicle group) were sacrificed at 24 h after injection. Myocardium and cortices tissues were quickly acquired and stored at -80 °C. Then small pieces of tissues (about 50 mg) were homogenated in 500 µL of PBS (pH 7.4). H2S concentration of the homogenate was determined by HPLC analysis described above. The protein content of tissue was measured by the bicinchoninic acid (BCA) method using a BCA Protein Assay Kit (Pierce, IL, USA). The H2S content of samples were quantified by protein content.
Rat CA/CPR Model
Sprague-Dawley rats were anesthetized and heparinized (0.2 mL, I.P.). Then the CA/CPR rat model was established by the previous method  (described in Supporting Information). Rats were randomized into three groups: (1) sham (n = 6), which were subjected to the same procedure as the other groups except for CA/CPR; (2) Control group (n = 6), which underwent 5 min of CA and received injection of same volume of saline; (3) [email protected] group (n = 6), which underwent 5 min of CA and received injection of [email protected] (10 mg/kg). Drugs were immediately injected after successful resuscitation. At 24 h after reperfusion, the myocardium and cortices tissues were quickly acquired and stored for subsequent experimental analysis.
Quantitative Assessment of Neutrophil Accumulation
Heart and brain tissues were assessed for the myeloperoxidase (MPO) activity as a marker of neutrophil accumulation. Tissues were homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mM K3PO4 buffer (pH 7.0) and centrifuged for 30 min (20, 000 g, 4 °C). Supernatant was allowed to react with tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2, and the change in absorbance was measured by spectrophotometry at 650 nm. The MPO activity was defined as the quantity of enzyme degrading 1 mmol of hydrogen peroxide per min at 37 °C and expressed in milliunits per milligram protein.
Antioxidant Enzyme Activities
A total of 50 mg heart or brain tissue was homogenized in a 50 mM ice-cold potassium phosphate buffer (pH 6.8). Superoxide dismutase (SOD) activity, catalase (CAT) activity and the malonydialdehyde (MDA) levels were determined by the previously reported method . The activity of SOD and CAT, and the levels of MDA were all standardized by protein content, determined using a bicinchoninic acid (BCA) protein assay kit (Beytime Institute of Biotechnology, Nantong, China).
Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assay
Acquired heart and brain tissues were stained with H&E, followed by a TUNEL assay: the cell nuclei were stained with 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) color development kits (Roche, Basil, CH) in accordance with the manufacturers’ instructions. The cell nuclei that stained green were defined as TUNEL-positive nuclei and were monitored using a Nikon invert fluorescence microscope. The proportion of TUNEL positive nuclei per 500 nuclei was quantified at a 200⋅ magnification.
Western Blot Assay
A piece of ischemic brain or heart tissue was homogenized by a rotor-stator homogenizer in ice-cold RIPA buffer (Pierce, Pittsburgh, PA, USA), and incubated at 4 °C overnight. After boiling with loading buffer (Fermentas, Glen Burnie, MD, USA), denatured proteins were separated in SDS PAGE gel, and transferred onto PVDF membrane. The membrane was blocked with 5% nonfat milk, followed by incubation with primary antibody of Bcl-2, BAX and Caspase-3 (Abcam, Cambridge, MA, USA) at 4 °C overnight. HRP-conjugated secondary antibody (Kangchen Bio-tech, Beijing, China) was used to incubate the membrane for another 2 h. SuperSignal West Pico Chemiluminescent Substrate (Pierce, Pittsburgh, PA, USA) was poured on the membrane to develop the band captured by FluorChem Image System (Alpha Innotech, Santa Clara, CA, USA).
Evaluation of Heart and Cerebral Function
Repeated CA/CPR protocol (n = 6), and cardiac and cerebral function were evaluated at 24 h after CPR and drug injection. Cerebral function was evaluated by neurological deficit scale (NDS) and balance beam test as to the previous methods [31, 32]. After which, rats were anesthetized for transthoracic echocardiography assessment using the Philips IE 33 system and a 12 − 4 MHz linear transducer (S12-4, Philips, AMS, NED). Stroke volume, ejection fraction (EF) and fractional shortening (FS) were derived to evaluate cardiac function, which were performed by skilled observer blindly. Repeated the CA/CPR protocols and kept the rats for 30 d, and survival of the rats were compared between the [email protected] and Vehicle groups (n = 30).
All statistics were performed using SPSS Statistics Base 17.0 for Windows. Continuous data were expressed as mean ± standard errors (SEM). One-way analysis of variance (ANOVA) was used to examine statistical comparisons between groups. The significant difference between two groups was analyzed by Student’s t test. Survival condition was analyzed using the Kaplan–Meier method. A value of P < 0.05 was considered to be significant. All authors had full access to, and take full responsibility for the integrity of the data.