Cytotoxicity of zno nanoparticles

Material properties that control the cytotoxicity of ZnO nanoparticles Supported by NSF grants CBET nano EHS 1134468 and EAGER DMR-1137419 Alex Punnoose, Jordan Chess, Catherine Anders, John Rassmussen, Denise Wingett, Kelsey Dodge and Katie Rainey, Boise State University, ID. and Jeffery Greenwood and Robert Tanguay Oregon State University, OR Preferential cytotoxicity of ZnO NP to cancer cells Reported preferential killing of Jurkat and Hut-78 cancer cells by 8nm ZnO NP without any measurable effect on normal T and B cells: 1. A. Punnoose, K. M. Reddy and D. Wingett, US Patent No: 8,187,638, Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles. 2. Reddy, et al., Selective toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Applied Physics Letters, 2007, 90, p. 213902. 3. Hanley, et al, Preferential killing of cancer cells and activated human T cells using zinc oxide nanoparticles. Nanotechnology, 2008. 19: p. 295103. 4. Hanley, C., et al., Nanoscale Research Letters, 2009, 4: p. 1409. 5. Thurber, et al, Nanotoxicology 2012, 6: p. 440-452. NP toxicity to cancer cells 0% 20% 40% 60% 80% 100% 0 0.1 0.2 0.3 0.4 0.5 [NP] in mM % viable cells Jurkat Hut-78 normal T cells ZnO NP toxicity: Conflicting reports Preferential cytotoxicity of ZnO NP to cancer cells was observed by other groups: 1. Ostrovsky, S., et al., Selective cytotoxic effect of ZnO nanoparticles on glioma cells. Nano Research, 2009. 2: p. 882. 2. Sasidharan, A., et al., Rapid dissolution of ZnO nanocrystals in acidic cancer microenvironment leading to preferential apoptosis. Nanoscale, 2011. 3: p. 3657. 3. M. Premanathan, et al., Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation. Nanomedicine: NBM, 2011. 7: p. 184. 4. L. Taccola, et al, Zinc oxide nanoparticles as selective killers of proliferating cells. International Journal of Nanomedicine, 2011. 6: p. 1129. Strong toxic response of ZnO NP to normal healthy cells, snails and embryonic zebrafish: 1. George, S., et al., Use of a Rapid Cytotoxicity Screening Approach To Engineer a Safer Zinc Oxide Nanoparticle through Iron Doping. ACS Nano, 2010. 4: p. 15-29. 2. Brunner, T.J., et al., In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environmental science technology, 2006. 40: p. 4374. 3. Croteau, M.N., et al., A novel approach reveals that zinc oxide nanoparticles are bioavailable and toxic after dietary exposures. Nanotoxicology, 2010. 5: p. 79. 4. Bai, W., et al., Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism Journal of Nanoparticle Research, 2010. 12: p. 1645. 5. Sun, J., et al., Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles in human cardiac microvascular endothelial cells, Cell Biol Toxicol., 2011. 27: p. 333. What makes identical NP? ZnO-I: Forced hydrolysis of Zn acetate dehydrate using diethylene glycol (DEG) as the solvent. ZnO-II: Forced hydrolysis of Zn acetate dehydrate using denatured ethanol, using LiOH to maintain correct pH; aged in n-Heptane. X-ray diffraction shows single phase wurtzite ZnO with average crystallite size of 8 ± 2 nm for both ZnO-I and ZnO-II. Lattice parameters were around a=3.25± 0.0003 Å and c=5.21± 0.003 Å. Journal of Applied Physics 113, 17C302 (2013). Physical Review B 82, 054419 (2010). Morphology - ZP TEM confirmed crystallite size ~8nm. Energy Dispersive Spectroscopy and X-ray Photoelectron Spectroscopy confirmed elemental composition. Zeta potential: ZnO-I: 40mV ZnO-II: 12mV Zeta Potential 5 6 7 8 9 10 11 12 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 Zeta Potential (mV) pH ZnO 1 4 nm ZnO 1 8 nm ZnO 2 5 nm ZnO 2 7 nm ZnO – I show larger ZP in the +30 to 50m V ZnO – II NP show much lower ZP < +20 mV FTIR Spectroscopy: Surface structure ZnO-I has surface adsorbed DEG layer Both show surface adsorbed groups related to acetate and significant hydroxyl bands. Optical/PL properties of ZnO NP 350 400 450 500 550 Normalized PL Intensity (a.u.) Wavelength (nm) As-prepared ZnO-II 3.50282 2.51012 ZnO-I Normalized PL Intensity (a.u.) As-prepared The PL spectra recorded at 10K using a 325 nm He:Cd laser. Both samples show the UV emission band at ~3.37 eV due to near band- edge transition of wide band gap ZnO NPs (free exciton recombination). ZnO-II NP show large green emission near 500nm. Large number of defects (oxygen vacancies/ interstitials)? ZnO NP size (nm) a (Å) c (Å) vol (ų) NC021 12.892 3.2547 5.2169 47.859 NC040 6.7066 3.2602 5.2231 48.077 NC042 9.9511 3.2595 5.2222 48.051 NC043 9.3459 3.2606 5.2265 48.121 NC044 8.2834 3.2606 5.2262 48.119 NC045 9.9555 3.2602 5.2236 48.083 NT010 10.916 3.2586 5.2185 47.989 NT114 9.3787 3.2582 5.2208 47.999 QC016 9.1523 3.2586 5.2202 48.006 QC017 10.463 3.2549 5.2153 47.849 QC019 8.4146 3.2576 5.225 48.019 Modified physico-chemical properties by varying hydrolysis ratio, reaction temperature, centrifuge speed, number of washing cycles, etc Toxicity of modified ZnO NP to CD4 T cells and Hut 78 cancer cells Trend lines depicting toxicity levels of nanoparticles. Blue=Hut 78 and Red=CD4 T cells. Y- axis is viability detected by PI via FACS and x-axis is mM concentration of nanoparticle. [...]...Role of surface charge/ZP of ZnO NP Role of hydrodynamic size of ZnO NP Role of catalytic activity of ZnO NP • Rhodamine B as a model dye to evaluate the catalytic activity using absorbance peak at 553nm • The photocatalytic rate constant k, kt = ln(C0/C) (where t = time, C0 = initial concentration of RhB molecules, and C = concentration after UV irradiation)... UV) 0.25 ZnO NP in Dark, no UV Arb units 0.20 RhB Bulk ZnO NC021 QC016 NC044 NC045 0.15 0.10 0.05 0.00 450 500 550 Wavelength (nm) 600 Catalytic activity of ZnO NP in dark 0.60 0.55 Role of catalytic activity of ZnO NP on Hut-78 Cancer cells 0.50 IC50 0.45 0.40 0.35 0.30 Using PI and FACS Using Alamar Blue 0.25 0.00030 0.00035 0.00040 Catalytic rate constant k (in dark) 0.00045 Conclusions • ZnO NP display... 0.45 0.38 23.3 1395 Catalytic activity of ZnO NP in dark 0.05 0.04 RhB NT010 QC017 NT114 NC042 Bulk NT034 NC021 NC045 NC044 QC019 QC016 Water 0.03 0.02 lnC/C0 0.01 0.00 -0.01 -0.02 -0.03 -0.04 -0.05 0 100 200 300 Time (min) 400 500 Zeta Potential ZnO – II NP show much lower ZP < +20 mV 50 ZnO 1 4 nm ZnO 1 8 nm ZnO 2 5 nm ZnO 2 7 nm 40 30 20 Zeta Potential (mV) ZnO – I show larger ZP in the +30 to 50m... experiments Journal of Applied Physics 113, 17B504 (2013) Catalytic activity of ZnO NP: UV vs Dark 0.22 0.00042 0.20 0.00040 0.18 0.00038 0.16 k in dark k in light 0.00036 0.14 0.12 0.00034 0.10 0.00032 0.08 0.00030 0.06 0.00028 0.04 Samples QC017 QC016 QC019 NT114 NC045 NC044 NC043 NC042 NC040 NC021 0.00026 Rate constant in light/UV 0.24 0.00044 Rate constant in dark 0.00046 Catalytic activity of ZnO NP in... reaction conditions and parameters significantly influence the physicochemical properties and cytotoxicity • Surface structure and defects plays the most important role in cytotoxicity – FTIR, ZP, PL are important to compare samples • Ability to tailor the critical materials/surface properties will allow the development of design rules for eliminating unwanted toxicity as well as to tailor cell specific toxicity... 1 4 nm ZnO 1 8 nm ZnO 2 5 nm ZnO 2 7 nm 40 30 20 Zeta Potential (mV) ZnO – I show larger ZP in the +30 to 50m V 10 0 -10 -20 -30 -40 -50 -60 5 6 7 8 9 pH 10 11 12 0.60 Hut 78 cancer cells and ZnO NP IC50 (mM of ZnO NP) 0.55 0.50 Using PI and FACS Using Alamar Blue 0.45 0.40 0.35 0.30 0.25 0.20 0.02 0.04 0.06 0.08 0.10 0.12 Rate constant k (using RhB) 0.14 . FACS and x-axis is mM concentration of nanoparticle. Role of surface charge/ZP of ZnO NP Role of hydrodynamic size of ZnO NP Role of catalytic activity of ZnO NP • Rhodamine B as a model dye. potential: ZnO- I: 40mV ZnO- II: 12mV Zeta Potential 5 6 7 8 9 10 11 12 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 Zeta Potential (mV) pH ZnO 1 4 nm ZnO 1 8 nm ZnO 2 5 nm ZnO 2 7 nm ZnO – I show. cells ZnO NP toxicity: Conflicting reports Preferential cytotoxicity of ZnO NP to cancer cells was observed by other groups: 1. Ostrovsky, S., et al., Selective cytotoxic effect of ZnO nanoparticles