Defect Engineering of ZnO Nanoparticles for Bioimaging Applications.

ZnO Nanoparticle Synthesis

The new synthesis procedure for producing nZnO was adapted from previous procedures with various modifications. 26 Zinc acetate dihydrate was used as the zinc source and diethylene glycol (DEG) as the solvent. To control the defects in the wet chemical synthesis, three parameters were varied; the amount of polyvinylpyrrolidone (PVP), water, and annealing temperature. The solution was created first by the simultaneous addition of PVP (molecular weight 58k) and zinc acetate, followed by the addition of DEG and initially heated to 80° C. Various amounts of water were then added, the temperature brought up to 150° C, and then held for 75 minutes. Once the solution cooled to room temperature, the samples were centrifuged at 41,410 X g to remove the DEG and subsequently washed with absolute ethanol. The pellet was dried overnight at 60° C and then annealed in air at the various temperatures indicated. Characterization. The nZnO samples were characterized using several techniques. X-ray diffraction (XRD) spectra were collected using a Rigaku Miniflex 600 X-ray diffractometer and used to determine the crystal phase, lattice parameters and to estimate the average crystal size using Rigaku PDXL software version 1.8.0.3.

Thermal gravimetric analysis

(TGA) was performed using a Netzsch STA449F1 TGA-DSC in order to define the ideal temperature and time to remove retained chemical species from the synthesis procedure.

Transmission electron microscopy

(TEM) was utilized to obtain both average crystal size distributions and determine the NP morphology using a JEOL JEM-2100 HR analytical transmission electron microscope. To obtain atomic concentrations and evaluate sample purity, both X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were utilized. A Physical Electronics Versaprobe system with a monochromated Al K α X-ray source was used to obtain the XPS spectra and a Bruker Tensor 27 spectrometer was used for FTIR spectra collection. FTIR pellets were prepared by grinding 1.5 mg of the NP sample with 0.200 g of spectroscopic grade KBr and subsequently pressed for 4 minutes with 8 tons of pressure. A Malvern Zetasizer NanoZS was used to collect dynamic light scattering (DLS) measurements by using a concentration of ZnO NPs of 0.5 mg/mL in nanopure water. For inductively coupled plasma mass spectrometry (ICP-MS) experiments, the nZnO were dissolved in 25 mL of highly pure 2% nitric acid solution and analyzed by a Thermo-electron X-series II quadrupole ICP-MS. The samples were analyzed for various metal contaminations such as iron, cobalt and nickel against the total zinc content. For Raman and photoluminescence (PL) experiments, the nZnO were mounted onto a silicon oxide wafer and the spectra were measured in backscattering geometry using a Jobin Yvon T64000 0.64m triple spectrometer equipped with a liquid nitrogen cooled multichannel CCD detector. Spectra were recorded at 10 K using a variable temperature closed cycle helium cryostat. Ultraviolet light (325 nm line of He-Cd laser) was used for excitation. ZnO Nanoparticle and Live Cell Imaging. Confocal microscopy was utilized to image both the nZnO and T47D cells treated with nZnO. Because this new method of nZnO synthesis achieves a lower band gap of ~3.1 eV, a 405 nm laser was able to be used as an excitation source and nZnO fluorescence monitored. For live cell imaging experiments, T47D (ATCC HTB-133), a cell line derived from ductal breast carcinoma (ATCC; Manassas, VA) was maintained in log phase and cultured in RPMI 1640 media following ATCC recommendations. Prior to imaging experiments, the cells were washed and resuspended in a custom made RPMI 1640 media free of HEPES, phenol red and phosphate (PPH free RPMI 1640; Thermo Fisher Scientific; Grand Island, NY) to prevent increased NP dissolution, remove fluorescent background signal and reduce the chemical transformation of the NPs, respectively. 10 , 27 The cells were then transferred to Nunc Lab-Tek II Chambered cell culture slides (ThermoFisher; Waltham, MA). Following an overnight incubation to ensure cell adhesion, nZnO were added at a final concentration of 30 μg/mL and further incubated to allow for nZnO-cell interactions. Control (nZnO-free) cells were cultured in the same manner to assess auto-fluorescence generated using the 405 nm laser to excite the nZnO. Prior to imaging, live cells were stained with the plasma membrane stain Cell Mask Orange (Invitrogen; Carlsbad, CA). In brief, stain was added to culture media for at a final concentration of 5 µg/ml. All confocal images were acquired with a Zeiss 510 LSM system with the Zeiss Axiovert Observer Z1 inverted microscope and ZEN 2009 Imaging software (Carl Zeiss, Inc.; Thornwood, NY) utilizing Diode (405 nm) and HeNe (543) lasers as excitation sources; a Plan-Apochromat 20x/NA 0.8, Fluar 40x/NA 1.3 oil, or α-Plan Fluar 100x/NA 1.44 oil objective; and band-pass BP filters of 420–480 nm and 550–647 nm to image the nZnO particles and lipid layer, respectively. The pinhole was set to 0.95 airy units correlating to a 0.6 µm section. Viability. Jurkat T cells (ATCC TIB-152) were cultured in log phase using RPMI 1640 media supplemented with 10% FBS (fetal bovine serum), 1% penicillin/streptomycin and 2 mM L-glutamine per ATCC (American Type Culture Collection) recommendations. For viability assessments, the cells were first washed with PPH free RPMI 1640 and then seeded at a concentration of 2.5 × 10 5 cells/mL using the same media in a 96 well plate. A stock of nZnO was prepared at 4.07 mg/mL (50 mM) and sonicated for 10 minutes. The stock solution was added to fresh cellular media to achieve a final nZnO concentration of 1.63 mg/mL (20 mM) and sonicated for an additional 10 minutes. The cells were then treated at the indicated concentrations and incubated at 37° C and 5% CO 2 for 48 hours. Cell viability was assessed using the Alamar Blue metabolic assay where Alamar blue was added to the wells at a final concentration of 10% 44 hours post treatment and incubated for an additional 4 hours. The fluorescence intensity measurements were performed using a Biotek Synergy MX plate reader using an excitation/emission of 530/590 nm.