Cellular internalization studies of semiconductor nanoparticles in bacteria
EMSL Project ID
21600
Abstract
When reduced to the nanoscale regime, many benign materials have been reported to develop toxicity. This is a potential public concern in relation to the use of nanomaterials in biomedical applications and their increased presence in the environment with the fast development of nanotechnology. Nanoscale metal oxide semiconductors such as ZnO and SnO2 with band gap energies in the 3-4 eV range have attractive optical and electronic properties, however, a detailed investigation of their cellular internalization and toxicity has yet to be conducted. The PI has extensive experience in the controlled synthesis and characterization of pure and doped ZnO and SnO2 nanoparticles and is making significant progress in improving their ferromagnetic properties. The PI, Dr. A. Punnoose, will investigate the cellular internalization and toxicity of pure and transition metal doped ZnO and SnO2 nanoparticles using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as model systems for gram-negative and gram-positive bacterial systems respectively. Cellular uptake will be investigated using detailed transmission electron microscopy (TEM) studies along with elemental mapping using the scanning TEM option in order to determine the nanoparticle concentration within the cells. The bacterial systems are relatively simple experimental systems and their exponential growth characteristics allow very simple methods to monitor the effect of toxic environments. The bacterial systems are also very convenient for detailed transmission electron microscopy studies with relatively simple sample preparation procedure. The purpose of this study is to primarily investigate the internalization of nanoparticles - Can the nanoparticles enter the bacteria cells? How is this process affected by the nanoparticle's size, nature of the surfactant layers, type of material system (ZnO versus SnO2), and type of bacterial system?. For TEM experiments, the bacteria will be chemically fixed prior to spotting them on the grids. To do this, the bacteria will be mixed in a 2.5% glutaraldehyde solution for a few hours. It will then be washed to remove the fixative and then spot the cells on the copper grids with formvar and carbon coating and dried in air before mounting on the TEM sample holder. The goals of the TEM studies are to investigate (i) changes in the cell structure, especially cell walls and cell morphology, (ii) location of the internalized nanoparticles, and (iii) concentration of internalized nanoparticles per cell. All these will be investigated as a function of nanoparticle size, material, surfactant coating and bacterial system. However, to estimate the nanoparticle uptake per cell, detailed analysis using the scanning TEM/EDX option is proposed. This will be required because the actual number of nanoparticles present in the vesicles of the bacterial cells is not easy to estimate from TEM images because of the possible clustering effect. Another very useful tool to investigate cellular internalization is a field emission scanning electron microscope (FE-SEM) available at PNNL. In scanning electron microscopy, morphological changes can be investigated using secondary electron imaging (SEI) while backscattered electron imaging (BEI) will provide information about the deeper interior of the cells where nanoparticles are more probably collected.
Project Details
Project type
Exploratory Research
Start Date
2006-08-09
End Date
2007-10-01
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members