Materials
The lipid components used in the formation of NPs included 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol (Chol), which were all purchased from Corden Pharma Inc (Liestal, Switzerland). IMTP (CSTSMLKAC, cyclic form, Mw = 943.2 Da) and DSPE-PEG2000-IMTP were synthesized and purified by Xi’an ruixi Biological Technology Co., Ltd (Shanxi, China). Iron oxide nanoparticles (10 nm) with oleic acid (OA- Fe3O4) were purchased from Ocean Nano Technology Co., Ltd (Springdale, AR, USA). PFH was obtained from J&K Scientific Ltd (Beijing, China). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was provided from Beyotime Biotechnology (Shanghai, China). 4′, 6-Diamidino-2-phenylindole (DAPI) and 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Agarose was received from Invitrogen (Carlsbad, California, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were purchased from Gibco Co. (Carlsbad, CA, USA). Penicillin–streptomycin was obtained from Boster Biological Technology Co., Ltd (Wuhan, China). Cell Counting Kit 8 (CCK-8) was provided by Dojindo Molecular Technology (Tokyo, Japan). Hydrogen peroxide (H2O2), trichloromethane (CHCl3), and other analytical grade reagents were purchased from Chongqing Chuandong chemicals (Chongqing, China).
Synthesis of NPs
10 mg of DPPC, 4 mg of DSPE-PEG2000-IMTP, 3 mg of cholesterol, and 200 ul OA-Fe3O4 (5 mg/ml) were dissolved into 5 ml of CHCl3, and then the solution was transferred to a round flask to form lipid films by rotary evaporation (Yarong Inc, Shanghai, China). Two hours later, the lipid film that appeared at the bottom of the flask was hydrated by 4 ml of double-distilled water. After adding 200 μl of PFH, the suspension was emulsified in an ice-water bath using a sonicator (Sonics & Materials Inc., USA) at 125 W for 8 min (5 s on and 5 s off). To remove free lipids and excess reactants, the nano-emulsion was centrifuged at 8,000 rpm for 5 min at 4 ℃ and repeated thrice. Finally, IMTP-Fe3O4-PFH NPs were obtained and then stored at 4 ℃ for later use.
The preparation method of non-targeted NPs (Fe3O4-PFH NPs) was the same as described above, except that DSPE-PEG2000 was used instead of DSPE-PEG2000-IMTP. Additionally, IMTP-PFH NPs were acquired as the ferric blank control without OA-Fe3O4 loading, and similarly, PFH was not added as IMTP-Fe3O4 NPs.
To prepare various fluorescent NPs, the fluorescent dye DiI or DiR (5 μl) was added to the lipid solution for rotary evaporation together, and tinfoil was used to prevent light exposure.
Characterization
The size, morphology, and dispersion of the IMTP-Fe3O4-PFH NPs were observed by the light microscope (Nikon, Japan) after dilution 100 times. A transmission electron microscope (TEM) (H-7600; Hitachi, Tokyo, Japan) was used to further ratify the IMTP-Fe3O4-PFH NPs morphology. The mean particle size and zeta potential of the different NPs were detected by Zetasizer NANO ZS system (Malvern Instruments Ltd., Malvern, UK). The concentration of Fe was measured with an atomic absorption spectrometer (Hitachi model Z-5000, Hitachi Ltd., Tokyo, Japan). The encapsulation efficiency and loading capacity of Fe3O4 were calculated by the following equation: Encapsulation efficiency (%) = mass of Fe3O4/total Fe3O4 × 100%; Loading content (%) = mass of Fe3O4/total liposomes × 100%. To test the stability of IMTP-Fe3O4-PFH NPs, the size of NPs was measured at each indicated time point (0, 1, 3, 5, 7 d).
Cell culture and animal model establishment
Rat myocardial cells (H9C2) purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd (Shanghai, China), were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml) at 37 °C in a humidified incubator under normal conditions (5% CO2, 21% O2 and 74% N2). After being cultured for 2–3 days, cells were trypsinzed and subcultured.
The hypoxia injury in cells was established by chemical reagent H2O2 or subjected to hypoxia conditions. In a subset of experiments, H9C2 cells were placed in a 37 °C airtight box saturated with 94% N2, 5% CO2, 1% O2 for different durations. Besides, the medium was changed to serum-free DEME. In another subset of experiments, cells were insulted with different concentrations of H2O2 for 24 h. Through cell morphology observation and CCK-8 detection of cell vitality, the most suitable conditions were selected for subsequent experiments.
Male Sprague–Dawley (SD) rats (with a bodyweight of 220 to 270 g) were maintained in the Center of Experimental Animals at Chongqing Medical University (Chongqing, China). Rats underwent ligation of the left anterior descending (LAD) coronary artery to induce acute myocardial ischemia. Briefly, the rats were anesthetized with 1% pentobarbital sodium (40 mg/kg, administered intraperitoneally), and ventilated via a rodent ventilator (Shanghai Alcott Biotech co., Ltd. China). The LAD was ligatured with a 6/0 suture. Successful occlusion of LAD was confirmed by the presence of S-T segment elevation on the electrocardiogram (ECG) (Guangzhou sanrui electronics co. Ltd, China) and a color change in ventricular from fresh red to pale. After confirmation of successful molding, the muscles and skin were sutured layer-by-layer and disinfected the incision. Postoperative echocardiography and histological examination after two weeks were used to further verify the success of modeling, so as to ensure the consistency of the model in subsequent experiments.
In vitro cytotoxicity and in vivo biosafety assay of NPs
To evaluate the cytotoxicity of IMTP-Fe3O4-PFH NPs, H9C2 cells were seeded in a 96-well plate (0.5 × 105 cells per well) and cultured for 24 h. DMEM without FBS was used to prepare different concentrations of IMTP-Fe3O4-PFH NPs (0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml). After another 24 h co-incubation, cell viabilities were tested by the CCK-8 assay. Six replicates were set for each group.
To assess the in vivo toxicity of IMTP-Fe3O4-PFH NPs, twenty-five healthy SD rats were randomly divided into five groups as follows: control group, 1 d, 3 d, 7 d, and 14 d experimental group (n = 5 per group). Via tail vein injection, the control group was injected with saline (1 ml), and the experimental groups were all administrated with IMTP-Fe3O4-PFH NPs (1 ml, 5 mg/kg). During the whole period of the experiment, the abnormal reaction, death, and its occurrence time were observed and recorded. At different points in time, 3–5 ml of whole blood was collected from the heart, part of which was used for routine blood detection, and part of which was separated from serum for biochemical analysis. The routine blood test mainly contained red blood cell (RBC), white blood cell (WBC), platelet (PLT), hemoglobin (HGB), and mean corpuscular volume (MCV). The biochemical indexes included aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), urea (UREA), creatinine (CREA), and lactate dehydrogenase (LDH). At the end of the experiment, rats were sacrificed and major organs (heart, liver, spleen, lung, and kidney) were taken for histological analysis with hematoxylin and eosin stain (H&E).
In vitro targeting efficiency
According to the previous model establishment, respectively with low oxygen or H2O2 treatment, H9C2 cells were seeded in culture dishes specified for confocal laser scanning microscopy (CLSM) (Nikon, Japan) at a density of 1 × 104 cells per dish. After 24 h, cells were divided into 4 groups: hypoxia + IMTP-Fe3O4-PFH NPs, hypoxia + Fe3O4-PFH NPs, H2O2 + IMTP-Fe3O4-PFH NPs, H2O2 + Fe3O4-PFH NPs. All groups were mixed with 100 μl DiI-labeled NPs (1 mg/ml) for 0.5 h, 1 h, 2 h, and 4 h respectively. After co-incubation, cells were washed thrice with PBS, fixed with 4% paraformaldehyde for 10 min, and then incubated with DAPI for 5 min. After that, the dishes were washed with PBS three times and sent for CLSM while protected from light.
Ex vivo targeting ability and biodistribution
To study the targeting efficiency and biodistribution of the NPs, model rats were randomly divided into two groups (n = 3) and injected with DiR-labeled IMTP-Fe3O4-PFH NPs or Fe3O4-PFH NPs via the tail vein immediately after the operation. Ex vivo fluorescence imaging of the heart and other major organs, including lung, spleen, liver, and kidney were acquired at pre, 5, 10, 30 min, 1, 2, 4, and 8 h post-injection by a fluorescence system (CRi Inc, Woburn, MA, USA), and the relative fluorescence intensities were calculated.
Phase transition and US imaging in vitro and in vivo
To determine whether IMTP-Fe3O4-PFH NPs could exhibit a phase transition behavior and whether PFH acted in this process, the IMTP-Fe3O4-PFH NPs and IMTP-Fe3O4 NPs were exposed to LIFU (1.0 MHz, focal length of 1.5 cm, duty cycle of 50%, pulse-wave mode, Chongqing Medical University, China) at different acoustic intensity (from 1 to 4 W/cm2) and different times (from 1 to 4 min), respectively. After irradiation, optical microscopy images were collected, and US images both in B-mode and contrast-enhanced ultrasound (CEUS) mode were evaluated by a US system (Esaote MyLab90, Florence, Italy) with a high-frequency linear array probe (LA523, frequency of 12 MHz, mechanical index of 0.06). Images before LIFU irradiation were used as controls. The echo intensities of regions of interest (ROI) were calculated by an ultrasound imaging software (DFY-II; Institute of Ultrasound Imaging, Chongqing, China) [44, 45].
For in vivo imaging, rats were divided randomly into 2 groups (n = 3): IMTP-Fe3O4-PFH NPs group, and Fe3O4-PFH NPs group. The chamber size, wall thickness, and amplitude of wall motion were assessed in the short-axis view. Each group of rats underwent ligation of LAD and were injected with NPs immediately as the baseline. 10 min after injection, all rats were irradiated by LIFU for 3 min (3 W/cm2, 1.0 MHz, focal length of 1.5 cm, duty cycle of 50%, pulse-wave mode). B-mode and CEUS images of pre-operation, baseline, after 10 min injection, and after LIFU irradiation were recorded. Besides, the intensities of ROI were compared.
In vitro and in vivo PA imaging
PA imaging experiments were performed by a Vevo LAZR Photoacoustic Imaging System (VisualSonics Inc., Toronto, Canada). The maximum absorbance of NPs for PA imaging was scanned at different wavelengths ranging from 680 to 970 nm (interval = 5 nm), which was then used as a basis wavelength for subsequent imaging. The IMTP-Fe3O4-PFH NPs were diluted to a series of Fe concentration from 0.039 to 0.624 mg/ml and added in an agar gel mold to acquire the corresponding PA images. Finally, the PA intensities of each image were calculated using Vevo LAZR software.
As for PA imaging in vivo, the model rats were intravenously injected with IMTP-Fe3O4-PFH NPs and Fe3O4-PFH NPs, respectively. Then, PA images were acquired before the operation, immediately after injection (baseline), 10 min and 60 min after injection. Furthermore, the corresponding PA signal values were measured.
In vitro and in vivo MR imaging
All MR experiments were performed conducted on a 7.0 T micro-MRI System (Bruker PharmaScan, Germany). The IMTP-Fe3O4-PFH NPs were dispersed in agar at different concentrations (0–0.32 mM) of Fe, and placed in 2 ml Eppendorf tubes for T2-WI using the following parameters: repetition time (TR) = 3000 ms, echo time (TE) = 9 ms, field of vision (FOV) = 45 × 40 mm, matrix = 256 × 256, slice thickness = 1.500 mm. The signal intensity of ROI was analyzed by RadiAnt DICOM Viewer and T2 relaxivities were calculated from the slope of the linear plots of r2 relaxation rates versus Fe concentration.
In vivo, MR imaging was conducted in model rats divided into 3 groups (n = 3), namely IMTP-Fe3O4-PFH NPs group, Fe3O4-PFH NPs group, and IMTP-PFH NPs group. Cardio-respiratory artifacts caused by cardiac pulsatility and respiration were reduced by using ECG and respiratory gating system. T2*-WI was run (TR = 40 ms, TE = 3.5 ms, flip angle = 40°, FOV = 40 × 40 mm, matrix = 192 × 192, slice thickness = 1 mm). Each image was processed by Matlab (2019) to get pseudo-coloring and the corresponding signal intensity was measured. After imaging, the hearts were harvested for Prussian blue & 3,3′-diaminobenzidine tetrahydrochloride (DAB) staining.
Statistical analysis
All statistical analyses were performed with SPSS 20.0 software. Data were presented as mean ± standard deviation. One-way ANOVA and the Student’s t-test were utilized between two groups for statistical evaluation. P values < 0.05 were considered statistically significant.