Fluorescence Modified Chitosan-Coated Magnetic Nanoparticles for High-Efficient Cellular Imaging
Labeling of cells with nanoparticles for living detection is of interest to various biomedical applications. In this study, novel fluorescent/magnetic nanoparticles were prepared and used in high-efficient cellular imaging. The nanoparticles coated with the modified chitosan possessed a magnetic oxide core and a covalently attached fluorescent dye. We evaluated the feasibility and efficiency in labeling cancer cells (SMMC-7721) with the nanoparticles. The nanoparticles exhibited a high affinity to cells, which was demonstrated by flow cytometry and magnetic resonance imaging. The results showed that cell-labeling efficiency of the nanoparticles was dependent on the incubation time and nanoparticles’ concentration. The minimum detected number of labeled cells was around 104by using a clinical 1.5-T MRI imager. Fluorescence and transmission electron microscopy instruments were used to monitor the localization patterns of the magnetic nanoparticles in cells. These new magneto-fluorescent nanoagents have demonstrated the potential for future medical use.
KeywordsMagnetic nanoparticle Fluorescence Chitosan Magnetic resonance imaging
Magnetic iron oxide nanoparticles (MIONPs) have been extensively utilized for drug delivery, magnetic resonance imaging (MRI), hyperthermia techniques, cell separation, and tissue repair [1–5]. Especially, when used as a contrast agent for the MRI, MIONPs allow researchers and clinicians to enhance the tissue contrast of an area of interest by increasing the relaxation rate of water. Although native MIONPs appear to be the currently preferred cell-labeling materials, the relatively poor signal intensity of MIONPs on MRI limits their clinical utility. Hence, more efficient cellular-internalizing methods are highly preferable. Recent studies on the size effect [6, 7], surface chemistry [8, 9], targeting ligands , and assemblies of MIONPs under magnetic field  have been reported to improve the internalization of the contrast agent. However, the internalizing efficiency is still generally low as manifested by the requirement of a long-term incubation or a high concentration of particles with cells.
The stabilized MNPs in aqueous solutions are promising candidates for biomedical applications. One possible way is to encapsulate them with polymeric materials. Ideally, this polymeric material should be biocompatible and possess reactive functional groups for the further attachment of biomolecules. Chitosan is a natural poly-cationic polymer that has one amino group and two hydroxyl groups in the repeating hexosaminide residue. It is an ideal polymer in biological applications owing to their being hydrophilic, biocompatible, biodegradable, non-antigenic and nontoxic [12, 13]. In addition, chitosan is known to facilitate drug delivery across cellular barriers and transiently open the tight junctions between epithelial cells .
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 . 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.
Fe3O4 nanoparticles were synthesized by chemical coprecipitation of Molday. In typical synthesis, a mixture solution of FeCl3 and FeSO4 (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-Fe2O3, MNPs) by air at the temperature of 90°C. During this step, the initial black slurry turning into brown could be observed . 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.
Results and Discussion
Characterize of FITC-CS@MNPs
Chitosan, which has a positive zeta potential, can interact with negative domain of cell membranes by nonspecific electrostatic interactions [13, 14]. FITC-CS@MNPs, with their tiny size and positive surface charge, showed a high electrostatic affinity for the cell membrane. Cellular internalization was initiated by nonspecific interactions between nanoparticles and cell membranes. It was reported that A549 cell uptake of chitosan nanoparticles occurred predominantly by adsorptive endocytosis, mediated in part by clathrin, but not by passive diffusion or by fluid-phase endocytosis . Cellular uptake of N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles also was reported to internalize by adsorptive endocytosis . There were no reports of chitosan-specific receptors on cell membranes.
In Vitro Cell-Viability and Cytotoxicity Studies
A novel magnetic fluorescent nanoparticle was prepared by a simple synthesis method and used for high-efficient labeling cancer cell. The FITC-CS@MNPs described here could be efficiently internalized into SMMC-7721 because of their electrostatic interactions with the cell membrane. These labeled cells can be visualized in a clinical 1.5-T MRI imager with detectable cell numbers of about 104in vitro. Magnetic fluorescent nanoparticles serve both as magnetic resonance contrast agents for MRI and optical probes for intravital fluorescence microscopy. Cytotoxicity test demonstrated that the prepared FITC-CS@MNPs possessed a suitable property for biomedical application.
This research has been carried out under the financial grants from The National Natural Science Foundation of China (Nos. 60571031, 60501009, and 90406023) and The National Basic Research Program of China (Nos. 2006CB933206 and 2006CB705600).