Preparation and Characterization of FITC-Labeled Chitosan Nanoparticles
The synthesis of FITC-labeled chitosan was based on the reaction between the isothiocyanate group of FITC and the primary amino group of chitosan [24
]. The FITC of 20 mg in 20 ml dehydrated methanol was added to 20 ml 1% w/v chitosan (low molecular, Sigma-Aldrich.) in 0.1 M acetic acid solution. After 3 h of reaction in the dark at ambient temperature, the FITC-labeled chitosan (FITC-CS) was precipitated by raising the pH to 10 with 0.5 M NaOH. The unreacted FITC was washed with distilled water and separated by centrifuge until no fluorescence was detected in the suspernatant. The FITC-CS dissolved in 20 ml 0.1 M acetic acid was then dialyzed in 4 l of distilled water for 3 days under darkness, with water being replaced every day.
nanoparticles were synthesized by chemical coprecipitation of Molday. In typical synthesis, a mixture solution of FeCl3
(molar ratio 2:1) was prepared under N2
shielding and then enough ammonia aqueous solution was poured into it while violently stirring. The black precipitate was formed and washed several times with deionized water. The final magnetite nanoparticles were dispersed in deionized water with pH 3.0 and oxidized into more stable maghemite (γ-Fe2
, MNPs) by air at the temperature of 90°C. During this step, the initial black slurry turning into brown could be observed [25
]. After that, MNPs were coated with FITC-CS (FITC-CS@MNPs), and 4 ml of above FITC-CS acetic acid solution was added to 50 ml of MNPs solution. The mixture was stirred for 4 h and then washed by the above magnetic separation method to remove dissociative FITC-CS.
Characterization of Magnetic Particles
The magnetic measurements were carried out using a Vibrating Sample Magnetometer (VSM, Lakeshore 7407, USA). The zeta potentials of the particles were determined by Zeta Potential Analyzer (BECKMAN, Delsa 440SX, USA). The particle morphology and size of the samples were determined by transmission electronic microscopy (TEM, JEOL, JEM-200EX). The emission spectra were measured with a Hitachi FL4500. The emission absorption spectra were measured using a LS-55 spectrophotometer (PerkinElmer, USA).
Human hepatoma cell line, SMMC-7721, was provided by Shanghai Cellular Institute of China Scientific Academy. Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS), 100 μg/ml penicillin, and 100 μg/ml streptomycin. For control experiments, medium having no particle was used. The cells were incubated at 37°C in 5% CO2atmosphere and medium was replaced every other day.
Cellular Uptake Experiments
In the cell-uptake experiments, the cells were incubated with different concentrations of FITC-CS@MNPs suspension in medium for various incubation times. After indicated times, the cells were washed three times with 0.1 M PBS, then harvested by trypsinization, centrifuged, and resuspended in 0.1 M PBS or 0.5 ml of 1% agarose in Eppendorf tubes. Cellular uptake of FITC-CS@MNPs was determined semiquantitatively by the incorporated fluorescence intensity and MR functionalities, using a BD FACS Calibur flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and a clinical 1.5-T MRI System (Eclipse, Philips Medical Systems, The Netherlands) by using a 12.7-cm receive-only surface coil, respectively.
The fluorescence of NBD- labeled green marker compounds was measured with a 488-nm argon laser excitation and a 530/30 bandpass filter for emissions. The whole amounts of cell surface uptake level and the intracellular uptake level were qualified by converting to an average number of molecules per cell.
The sequence parameters for T1-weighted (T1 W) imaging was spin-echo repetition time 500 ms, echo time 17.9 ms; T2-weighted (T2 W) imaging was fast spin-echo repetition time 4000 ms; echo time 108 ms; echo train length 16; T2*-weighted (T2*W) imaging was gradient-echo repetition time 620 ms, echo time 15.7 ms; flip angle 35°. Images were obtained with a matrix size of 256 × 256—two measurements were acquired: section thickness of 2 mm; field of view of 10 × 10 cm. Region of interest for signal intensity measurement was 20 mm2. These tubes contained 5 × 102, 1 × 103, 5 × 103, 1 × 104,5 × 104,1 × 105labeled cells, respectively. Another two Eppendorf tubes containing 1 × 106unlabeled cells and distilled water were used.
Fluorescent and Transmission Electron Microscopy
After magnetic nanoparticles labeling, adhering cells were washed three times with 0.1 M PBS and then fixed with 2% glutaraldehyde buffered in 0.1 M PBS for 1 h at 4°C. The optical and fluorescent images were observed with an Axioskop 200 microscope equipped with a Coolsnap MP3.3 camera (Carl Zeiss, Germany).
For the samples of TEM, the cells were washed three times with 0.1 M PBS, then harvested by trypsinization, centrifuged, and fixed with 2% glutaraldehyde buffered in 0.1 M PBS for 1 h at 4°C. The cells were then post-fixed in 1% osmium tetroxide for 2 h at 4°C, washed again with PBS, dehydrated through a series of alcohol concentrations (20, 30, 40, 50, 60, 70%), and followed by further dehydration(90, 96, 100% and dry alcohol). The cells were finally treated with propylene oxide followed by 1:1 propylene oxide: resin for overnight to evaporate the propylene oxide. The cells were subsequently embedded in Araldite resin, and ultra-thin sections cut with glass knives were stained with lead nitrate, and viewed under a HITACHIH-600 electron microscope at 80 kV.
In Vitro Cell-Viability/Cytotoxicity Studies
To determine cell cytotoxicity/viability, the cells were plated at a density of 1 × 104cells/well in 96-well plates at 37°C in 5% CO2atmosphere. After 24 h of culture, the medium in the wells was replaced with the fresh medium containing nanoparticles in the concentration range of 0–123.52 μg/ml. After 12 h, the medium was removed and rinsed twice with medium, and then 20 μl of MTT (3,4,5-dimethylthiazol-yl-2,5-diphenyl tetrazolium, Sigma) dye solution (5 mg/ml in medium) was added to each well. After 4 h of incubation at 37°C, the medium was removed, and Formazan crystals were dissolved in 200 μl dimethylsulphoxide (DMSO) and quantified by measuring the absorbance of the solution at 570 nm by a microplate reader (Model 680, Bio-RAD). The spectrophotometer was calibrated to zero absorbance, using culture medium without cells. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated by [A]test/[A]control × 100, where [A]test is the absorbance of the test sample and [A]control is the absorbance of control sample.
Each experiment was repeated three times in duplicate. The results were presented as mean ± SD. Statistical significance was accepted at a level ofP < 0.05.