Synthesis and characterization of FMNPs
FMNPs were prepared using improved method based on our previous reported method . First, Fe3O4@Polystyrene (Fe3O4/PS) nanospheres were prepared according to Ref. , CdTe QDs were prepared according to our previous method , then CdTe QDs were modified to the surface of Fe3O4@Polystyrene (Fe3O4/PS) nanospheres, resultant CdTe QDs-covered MNPs were modified with silica shell using reverse microemulsion method, finally prepared FMNPs were washed with PBS (pH 7.4), isolated, and then saved for usage. Prepared FMNPs were characterized using scanning electron microscopy (SEM), high-resolution transmission electron microscopy (TEM, JEOL JEM2010, at 200 kV), the photoluminescence (PL) spectra (Perkin Elmer LS 55 spectrofluorimeter), and superconducting quantum interference device (SQUID) magnetometer (Quantum Design, PPMS-9T).
Observation of cells incubated with FMNPs
HEK293 cells were seeded on cover slips for overnight, and were treated with medium with FMNPs (50 μg/ml) for 3 h. Then, the cells were rinsed with distilled water and stained with Prussian blue according to protocol. The HEK 293 cells were not treated with medium with FMNPs as the control. Then, the samples were attached to glass plates using mounting medium, and then were observed under an optical microscope (Olympus IX71).
Fluorescent microscopy observation
HEK293 cells incubated with FMNPs (50 μg/ml) were collected, and fixed with 2.5% glutaraldehyde for 30 min, then, were incubated with 1 mM Hoechst 33258 in PBS (pH 7.4) for 5 min, and then were washed with PBS (pH 7.4) for three times, finally these cells were observed under fluorescence microscope (NIKON TS100-F).
Flow cytometry analysis
HEK293 cells were treated without or with 20 μg/ml of FMNPs for 24 h, and were collected. After washing with PBS (pH 7.4), these cells were fixed with 70% ethanol/PBS for 30 min on ice. Approximately, 4 × 105 cells were centrifuged and re-suspended with PBS (pH 7.4), and then were analyzed by Calibur Flow Cytometers (BD Biosciences, Sunnydale, CA), the number of cells labeled with FMNPs were counted on.
TEM observation of endocytosis course of FMNPs
HEK 293 cells were treated with 50 μg/ml FMNPs for 24 h. Then these cells were collected and fixed with 2.5% glutaraldehyde solution, and then embedded with epoxy resin, finally, these cells were made into the ultra-thin slices, and then observed with TEM.
Effects of FMNPs on HEK 293 cells viability and proliferation assay
Effects of FMNPs on proliferation and viability of HEK293 cells were analyzed using Cell Counting Kit-8 (CCK8) assay. HEK293 cells were cultured in the 96-well microplate at the concentration of 5000 cells per well and incubated in a humidified 5% CO2
balanced air incubator at 37°C for 24 h. Except for control wells, the remaining wells were added into medium with FMNPs, final concentrations were, respectively, 20, 50, and 100 μg/ml, then those cells were continued to culture for 1-4 days, then, the ODs were measured using the Thermo multiskan MK3 ELISA plate reader according to the protocol of CCK8 assay kit, and calculated the survival rate of cells. The survival rate of cells can be calculated by the following equation:
Detection of cell adhesion ability
Six-well plates were coated with fibrinogen (5 μg/ml) and vitronectin (1.5 μg/ml) in Dulbecco's Phosphate Buffered Saline (DPBS). HEK293 cells were harvested, washed for three times with serum-free minimal essential medium with Eargle's salt, and resuspended in attachment solution (calcium- and magnesium-free Hanks' balanced salt solution, 20 mM HEPES, 1 mg/ml heat-inactivated BSA, 1 mM CaCl2
and 1 mM MgCl2
). Except for the control cells without treated with FMNPs, each well was added 1 × 104
HEK 293 cells and cultured for 1-4 days with FMNPs at different concentrations: 20, 50, and 100 μg/ml, unattached cells were washed with Hanks' balanced salt solution. The numbers of remaining attached cells after centrifugation were quantified spectrophotometrically at 405 nm for three times [55
Western blotting analysis of adhesive proteins
HEK293 cells were, respectively, incubated with FMNPs (20, 50, and 100 μg/ml) for 4 days, then they were collected and lysed in protein lysis buffer. Equal amounts of sample lysates were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was blocked with 0.1% BSA in Tris-Buffered Saline Tween-20 (TBST) buffer, and incubated overnight at 4°C with specific primary antibodies such as anti-fibronectin monoclonal antibody, anti-cyclin D3 antibody, anti-laminin antibody, anti-FAK antibody, and anti-β-actin antibody. Subsequently, the membrane was washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies. Enhanced chemiluminescence kits were used (Amersham, ECL kits) .
Effects of FMNPs on mice
All animal experiments were performed in compliance with the local ethics committee. Kunming mice (female 28-30 g, 4-5 weeks old) were obtained from the Shanghai LAC Laboratory Animal Co. Ltd., Chinese Academy of Sciences (Shanghai, China) and housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) on a 12:12-h light/dark cycle. The mice were allowed to acclimate at this facility for 1 week before being used in the experiment. All mice were divided into three test groups with FMNPs (20, 60, and 100 μg), and one control group (0 μg), and each mouse was injected with the suspension with FMNPs via tail vein. Those mice were, respectively, killed at No. 1-, 7- and 30-day post-injection, their organs such as heart, liver, spleen, stomach, kidneys, lungs, and brain were collected immediately. Then, those organs were fixed with 10% formaldehyde, embedded in paraffin, were cut into 20-μm section, stained with hematoxylin and eosin, and were observed by light microscopy. Three mice without FMNPs treatment were used as control.
After mice treated with different doses of FMNPs (20, 60, and 100 μg) for 1, 7, and 30 days, the blood were, respectively, collected in potassium EDTA collection tubes using a standard saphenous vein blood collection technique and were analyzed for standard hematology markers: red blood cell count (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, and white blood cell count (WBC). In order to separate serum, blood samples were centrifuged twice at 3000 rpm for 10 min. Liver function was evaluated with serum levels of total bilirubin levels (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkalinephosphatase (ALP). Nephrotoxicity was determined by blood urea nitrogen (BUN) and creatinine (Cr). The enzyme of lactate dehydrogenase (LDH) was assayed for evaluating cardiac damage. Albumin (ALB) was assayed as one parameter of damage of tissue or inflammation. These parameters were all assayed using a Hitachi 7600 Automatic Biochemical Autoanalyzer.
Each experiment was repeated three times in duplicate. The results were presented as mean ± SD. Statistical differences were evaluated using the t test and considered significance at P< 0.05.