Functional iron nanoparticles for cell labeling and DNA separation

Magnetic-fluorescent nanoparticles can offer various applications such as imaging, labeling, separation, and therapy. Here we have functionalized magnetic nanoparticle (MNP) with fluorescent dyes such as acridine orange (ACO) and rhodamine-B (RHB) via chemical crosslinkers. The fluorescent MNPs were characterized by TEM, SQUID, and FTIR to prove the successful conjugation. Fluorescent spectroscopy, InCell analyzer and confocal microscopy confirmed that the ACO-MNP and RHB-MNP exhibited fluorescent property and cell-labeling ability. Furthermore, the functional MNP shows strong intracellular fluorescence when incubated with the 293T cells within 1 min. The cell viability tests for these fluorescent MNP on 293T cells shows low cytotoxicoty indicating the safeness of the nanoparticles. The multifunctional iron nanoparticles can offer a versatile platform for cell labeling and DNA separation. keywords:iron nanoparticles, fluorescence, cellular image, bioseparation. Introduction Magnetic–fluorescent composite nanoparticles combine the fluorescence, magnetic properties and nano-size, so they can be applied in bioseparation, imaging, sorting, drug delivery and cancer therapy. Functionalized magnetic nanoparticles can label selected targets with fluorescent signal in the presence of other suspended solids (Shu et al. 2010, Wang and Su 2011). Specific ligand modification on the magnetic nanoparticles surface allows rapid isolation (Song et al. 2011, Di et al. 2012), efficient labeling(Wang et al. 2012, Lehmann et al. 2010, Chen et al. 2012), and clear imaging (Xu et al. 2010, Ebrahiminezhad et al. 2013).. Acridine orange [3,6-bis(dimethyl) acridinium chloride] (ACO) is normally used to probe DNA/RNA structure in drug–DNA/RNA interactions. ACO has been used extensively for cell staining of DNA in apoptosis studies. It binds DNA via intercalation and stabilizes pigment–DNA complexes through charge neutralization with DNA backbone phosphate group. The binding of the intercalator to double-stranded DNA greatly enhances its fluorescence intensity and lifetime. The interaction of planar heterocyclic ACO with DNA includes three modes: (i) intercalating between stacked base pairs, thereby distorting the DNA backbone; (ii) as major or minor groove binders, causing little distortion of the DNA backbone; (iii) interaction with the external DNA double helix, which does not possess selectivity. The positive charge on ACO exocyclic amines exhibits favorable π-stacking interactions with DNA base pairs by mediating electrostatic attraction and hydrogen bonding interactions with DNA phosphate groups. Compared to conventional processes, the advantages of magnetic separation are attributed to its speed, accuracy, and simplicity. Additionally, they can be easily recovered and regenerated, even in the presence of colloidal contaminants. The problems of membrane fouling in microfiltration and pressure drops in chromatography can be reduced by using the magnetic separation. There are several crosslinking agents that can modify the surface property and functionalize the magnetic particles. For example, 1-Ethyl-3-dimethylaminopropyl carbodiimide (EDC) can activate carboxyl groups on the ligands for sequential conjugation with amino groups on MNP surfaces to create a zero-length amide bond. Glutaraldehyde can conjugate two amine-containing molecules in aqueous solutions by reductive amination. The reaction mechanism for this modification proceeds by forming two Schiff base linkages with two amines on ligands and MNP surfaces. The design of the composite nanoparticels, specifically how ACO affects the iron nanoparticles size, surface charge, fluorescence property and plasmid adsorption, is also not well understood. Very few papers have studied the fluorescence property and adsorption behaviors of ACO-modified magnetic nanoparticles. In this study, the adsorption of plasmid and salmon sperm DNAs onto surface-modified MNPs was systematically investigated by using Langmuir and Freundlich models. The ACO and rhodamine on nanoparticles was confirmed by FTIR. The fluorescent property of ACO-MNPs and their DNA complexes were characterized by using confocal microscopy and fluorescence spectroscopy. Materials and Methods Materials Acridine orange, rhodamine B, glutaraldehyde, KBr, hydroxylamine, o-phenanthroline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paramagnetic iron oxide nanoparticles (USPIO-101, NH2 modified; USPIO-102, COOH modified;) were purchased from Taiwan Advance Nanotech (Taoyuan, Taiwan). Plasmid EGFP-C3 (size 4.7kb) was obtained from Takara Bio (Shiga, Japan). The plasmid was amplified in Escherichia coli DH-5 alpha and purified using a purification kit (GeneMark, Taipei, Taiwan). Single strand DNAs from salmon milt was obtained from Maruha-Nichiro Foods Inc (Tokyo, Japan). The size of the salmon milt DNA ranges from 300 to 500bp. Purity of pDNA and ssDNA (OD260/OD280 ratio) was estimated higher than 1.8 by using the Nanodrop 1000 spectrophotometer. The water used in this study was freshly purified by a Milli-Q Gradient A10 system (Millipore, Molsheim, France). All reagents were used without further purification. Preparation of dye coated magnetic nanoparticles Surface modification of paramagnetic iron oxide nanoparticles was achieved by a coupling reaction between the amino groups of ligand and the amino groups of nanoparticles using glutaraldehyde, as a coupling reagent. Magnetic nanoparticles (1 mg) were mixed with 1 ml of a solution containing glutaraldehyde (10%), to pre-crosslink the particles and reacted at a constant vortex rate (of magnitude 4) for 1 h. The particles were washed three times with deionized water and are recovered using a magnetic separation system (Millipore). Acridine orange (ACO, 0.17%) was added to the nanoparticles with vortex for 2 hours to further crosslink the particles. The un-reacted ACO was removed by three wash with water. The ACO-MNP was collected and purified by the magnet stand. The stability of ACO-MNP in water could maintain for 1month. For all experiments, triplicate was performed in order to determine mean and standard deviation (SD). The iron content of MNP was quantified via modified o-phenanthroline method as described by Tamura et al (1974). Because MNP contains both ferrous iron (Fe2+) and ferric iron (Fe3+), all of ferric iron in the MNP shall be reduced from Fe3+ to Fe2+ by the use of an excess of hydroxylamine. Then the ferrous iron reacts with o-phenanthroline to form orange complexes. The absorbance of the complexes is measured by Epoch Spectrophotometer H4 (Biotek, Winooski, VT) at wavelength of 510 nm. MNP concentration is adjusted to 1mg/mL by the iron content determined by the calibration curve. Cell culture and MNP uptake analysis The cell lines were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). HEK-293T (BCRC 60019) was maintained in Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with 10% fetal bovine serum (FBS). Cells were incubated at 37°C in a 5% CO2 atmosphere and subcultured every 2~3 days using trypsin-EDTA. The cells were washed twice with 2-3 ml PBS buffer, then 1 ml trypsin was added and the cells were incubated for 2 min in order to trypsinize the cells monolayer. The cells were suspended in DMEM/FBS medium and the concentration of cells was determined by Multisizer 3. For MNP uptake experiments, cells were seeded on 48 well plates at a density of 5×10 4 cells/well i and allowed to grow for 48 hours n 48 well plates. The DNA/nanoparticle complex was prepared by mixing 1 μg of plasmid with 20 μg of MMP solution and allowed to stand at room temperature for 30 min. DMEM medium (0.2mL) containing DNA/MNP complexes was added to the 48-well plate at 37 °C for various incubation intervals. At the end of the incubation, cells were fixed by 0.2mL of 4% formaldehyde and analyzed by INCell Analyzer 1000 (GE Healthcare, Piscataway, NJ). This high-throughput analyzer is a bench top automated microscope designed for imaging cellular assays. The system contains a Nikon microscope, xenon lamps, a high-resolution CCD camera and a laser auto-focus device. Cells in 48 well plates were analyzed for fluorescence intensity and ACO positive percentage by the INCell Analyzer. The fluorescence at 455 nm in the nucleus stained by Hoechst 33342 was recorded after excitation at 350 nm. The fluorescence of ACO at 525 nm was quantitatively analyzed after excitation at 480 nm. To analyze the fluorescent images, the INCell Investigator was used to identify the viable cells (blue fluorescence) and ACO-positive cells (green fluorescence). The image was acquired in a portion (1/13th) of the view field by the 10× objective. Twenty views were analyzed to obtain the fluorescence intensity. Non-treated cells were analyzed in parallel as a control. The percentage of fluorescence positive cells is defined as follows: (the number of cells exhibiting both blue and green fluorescence/the number of cells exhibiting blue fluorescence) ×100%. The cell viability is defined as follows: (the number of nuclei in controlled cells/ the number of nuclei in treated cells) ×100%. Adsorption of pDNA or ssDNA by magnetic nanoparticles The MNP about 100μg was added to DNA solution (200μl) at various DNA concentrations containing 0.5 M NaCl in an eppendrof tube and incubated for 30 min. The MNPs were removed magnetically from DNA solution after 3 min. The amounts of DNA adsorbed on the magnetic nanoadsorbents were estimated from the mass balance of DNA in solution. The concentration of nucleic acids in solution can be readily calculated from absorbance at 260nm by Nano Drop Spectrophotometer (Thermo, Wilmington, DE). The adsorption experiments were repeated three times. Adsorption/desorption of pDNA or ssDNA by magnetic nanoparticles Equilibrium experiments of adsorption and desorption were performed in the following manner. The magnetic adsorbents were washed twice by using deionized water at room temperature. The magnetic adsorbent (10 μg) was transferred to microcentrifuge tubes and incubated with the DNA solution (containing 0.1M NaCl) for 30 min. After 3 min separation by using the magnet, the DNA content in the supernatant was analyzed by the spectrophotometer and the adsorbed DNA was calculated from the mass-balance equation. The DNA amount was measured at 260 nm using the spectrophotometer (Thermo, NanoDrop Technologies, Wilmington, DE, USA). The adsorption kinetics was determined using the following procedure. Magnetic nanoparticles were added to 0.01 ml of the DNA solution with different DNA concentrations. The DNA in the supernatant was determined spectrophotometically. A series of DNA concentrations was used to study MNP adsorption isotherm. The adsorption data were fitted by the two isotherm equations including Langmuir (Eq. 1) and Freundlich (Eq. 2) models employing the iterative fitting method of Sigmaplot (Version 10.0, Systat software, San Jose, CA).