Labeling TiO2 nanoparticles with dyes for optical fluorescence microscopy and determination of TiO2-DNA nanoconjugate stability.

4.

Experimental Nanoconjugate Preparation

All reagents were purchased from Sigma Aldrich unless otherwise specified. TiO 2 nanoparticles with mean diameter of around 5-6 nm were synthesized at Northwestern University’s Nanofabrication Core of NU-Center for Cancer Nanotechnology Excellence, applying low-temperature alkaline hydrolysis route, dialyzed, and stored in Na 2 HPO 4 buffer (10 mM) at 4°C. Surface coating with glycidyl isopropyl ether was performed as described previously.[ 9 ] Conjugation of single stranded 5’ carboxyl deoxythymidine modified DNA oligonucleotides (ttccttggatgtggt) (The Midland Certified Reagent Co.) to dopamine, and subsequent conjugation to TiO 2 nanoparticles was performed as described previously.[ 9 , 10 , 20 ] The oligonucleotides were also purchased with either tetramethylrhodamine (TAMRA) or Cy5 3’ end modification.

Alizarin Red S Surface Coating and UV-VIS Absorbance Spectrum Analysis

For determination of covalent surface modification, TiO 2 nanoparticles (5 μM) were dialyzed and stored in Na 2 HPO 4 buffer (10 mM) and mixed with Alizarin Red S (0.9 mM). The samples were then analyzed by the Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc.,) for absorbances ranging from 200-750 nm. Cell Culture and Treatment with Nanoparticles/Nanoconjugates All cell culture reagents were purchased from Mediatech Inc. unless otherwise specified. PC-3M metastatic prostate cancer cells were a gift from Dr. Raymond Bergan, Northwestern University. MCF-7/WS8 cells were obtained from American Type Culture Collection (ATCC). Both cell lines were maintained at 37°C with 5% CO 2 in RPMI 1640 media supplemented with fetal bovine serum (10%), L-glutamine (2 mM), HEPES (10 mM), penicillin (100 I.U./ml), streptomycin (100 μg/ml). MCF-7 cells were further supplemented with non-essential amino acids (1X), amphoterecin B (0.25 μg/ml), and insulin (0.1 mg/ml) (Sigma-Aldrich). PC-3M cells were further supplemented with G418 sulfate (0.15 mg/ml). For treatment, cells were washed with phosphate buffered saline solution (PBS) and placed in serum free RPMI 1640 for 1-2 hours. Then cells were treated with TiO 2 nanoparticles (333 nM) coated with or without ARS (60 μM), and/or ODN (160 μM) for one hour. After treatment, cells were washed with PBS, and then in glycine (200 mM, pH 4) (Sigma Aldrich). After more washing in PBS, the cells were further prepared as described below, depending on the technique performed. Transfection of cells was carried out using Superfect (Qiagen) according to manufacturer’s suggestions.

Flow Cytometry and Cell Sorting

Cells to be analyzed by flow cytometry were grown until approximately 60% confluence. After treatment and washing, cells were trypsinized, collected in FBS supplemented medium, and brought to a single cell suspension. To determine cell viability, cell samples were treated with DAPI (5 μg/ml) (Molecular Probes, Invitrogen) prior to analysis. Samples were then taken to the Northwestern University Flow Cytometry Core Facility of the Robert H. Lurie Cancer Center to be analyzed or sorted on the DakoCytomation MoFlo Flow Cytometer (Dako). Excitation lasers of 350 nm and 543 nm were used to excite DAPI and TAMRA/ARS, respectively. Debris was excluded based on the forward and side scatter characteristics of the cell populations. Analysis of flow cytometry data was performed using the FCS Express V3 program (De Novo Software).

Confocal Microscopy

For visualization by confocal microscopy, cells were cultured on glass coverslips. Cells at approximately 60% confluence were treated and washed as described above. The cells were then fixed in paraformaldehyde (4%), and stained with Hoechst 33343 (Molecular Probes, Invitrogen). After washing the cells were placed in anti-fade mounting medium (Molecular Probes, Invitrogen) and visualized using the LSM 510 UV Meta Microscope (Carl Zeiss, Inc.) at the Northwestern University Cell Imaging Facilities using 405 nm, 488 nm, and 543 nm lasers with bandpass filters of 420-480 nm, 505-530 nm, 560-615 nm, respectively. X-Ray Fluorescence Microscopy (XFM) Cells transfected with TiO 2 -DNA (TAMRA) nanoconjugates were sorted for the presence of TAMRA, seeded on formvar coated gold EM grids (Electron Microscopy Sciences) and allowed to adhere. The samples were then fixed in cold methanol (-20°C), and stained with Hoechst 33342 dye. The samples were placed in anti-fade mounting medium between a glass coverslip and slide, and imaged by confocal microscopy. Next, the cells were washed in glycine and PBS, dehydrated in ethanol (100%), and allowed to air dry. Before XFM analysis, the presence of the cells was verified, and coordinates for their locations were obtained by the Leica DMXRE light microscope and a motorized x/y stage (Ludl Electronic Products). XFM was performed at the 2-ID-D beamline at the Advanced Photon Source at Argonne National Laboratories where an undulator source was used to create hard X-Rays with energies of 10 keV and focused using Fresnel zone plate optics. Emitted X-Ray fluorescence was detected using an energy dispersive germanium detector (LEGe Detector, Canberra). Elemental quantification and localizations were calculated using the MAPS program.[ 50 ]

Alizarin Red S Surface Coating and UV-VIS Absorbance Spectrum Analysis

For determination of covalent surface modification, TiO 2 nanoparticles (5 μM) were dialyzed and stored in Na 2 HPO 4 buffer (10 mM) and mixed with Alizarin Red S (0.9 mM). The samples were then analyzed by the Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc.,) for absorbances ranging from 200-750 nm.

Supplementary Material Supplemental Figure 1 Multi-planar Comparison of Confocal Microscopy and XFM. ARS labeled TiO 2 -DNA nanoconjugates were transfected in PC3-M cells. Confocal Microscopy reveals in one plane the presence of ARS labeled nanoconjugates within the nucleus (1 top row) and another plane reveals localization in the cytoplasm (2 middle row). XFM (bottom) reveals two strong intracellular titanium signals (1,2), as well as several other smaller aggregates. Comparison suggests that these overlap with the distributions visualized by XFM. The high phosphorus signal is due to the presence of the EM grid to which the cell had adhered.