Evaluation of the microbial growth response to inorganic nanoparticles
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In order to enhance the utilization of inorganic nanoparticles in biological systems, it is important to develop a fundamental understanding of the influence they have on cellular health and function. Experiments were conducted to test silica, silica/iron oxide, and gold nanoparticles for their effects on the growth and activity of Escherichia coli (E. coli). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the morphology and quantify size distribution of the nanoparticles, respectively. TEM was also used to verify the interactions between composite iron oxide nanoparticles and E. coli. The results from DLS indicated that the inorganic nanoparticles formed small aggregates in the growth media. Growth studies measured the influence of the nanoparticles on cell proliferation at various concentrations, showing that the growth of E. coli in media containing the nanoparticles indicated no overt signs of toxicity.
KeywordsGold Nanoparticles Dynamic Light Scattering Dynamic Light Scattering Oxide Nanoparticles Luria Bertani
Research concerning the impact of inorganic nanoparticles on cellular health will enable new developments in nanobiotechnology to reach their fullest potential. An improved understanding of nanoparticles and biological cell interactions can lead to the development of new sensing, diagnostic, and treatment capabilities, such as improved targeted drug delivery, gene therapy, magnetic resonance imaging (MRI) contrast agents, and biological warfare agent detection [1, 2, 3, 4, 5, 6]. What is not certain about the production of these particles is whether they, alone, are toxic to cells in general.
Cytotoxicity is of major concern and will become increasingly so as the demand for nanoparticles grows with the development of more biological applications. Questions, such as how and if nanoparticles harm biological environments, how persistent they may be, and to what degree they affect other organisms including people are all concerns. It is known that nanoparticles can transfect cells; however, responses to nanoparticles inside and outside of cells are unknown. As nanoparticles become more common and widely produced, the chances of unplanned events leading to their dissemination and accumulation in the environment increase, and could lead to unforeseen changes to biological systems. In this study, E. coli served as a representation of how cells might respond to the presence of nanoparticles in their growth environment.
The goal of the research presented here is to investigate how nanoparticles interact with microbial cells, and what effect nanoparticles have on their growth process. Nanoparticles present a research challenge because little is known about how they behave in relation to microorganisms, particularly at the cellular level. The colloidal behavior of the inorganic nanoparticles in the microbial growth media was investigated to determine the stability of these systems in saline environments. Colloidal stability is an issue when dealing with biological environments due, in part, to the effect of salt on the nanoparticles. Agglomeration occurs causing sedimentation of the nanoparticles, thus limiting their interactions with growing cells, such as E. coli.
Three types of nanoparticles were used to conduct this study: silica, silica/iron oxide, and gold. The silica/iron oxide nanoparticles are important because of their magnetic properties. They could potentially be used for medical applications, such as MRI and targeted drug delivery applications. Another application would be to use them as biological sensors. Being that they are composites, the silica portion of the nanoparticle can be functionalized to attract various biological elements while the iron oxide portion can provide mobility under the presence of a magnetic field.
Gold nanoparticles are employed in multiple applications involving biological systems. Gold has exceptional binding properties, and this makes it attractive for attaching ligands to enhance various biomolecular interactions. These nanoparticles also exhibit an intense color in visible region for spectroscopic detection and also great contrast for electron microscopic imaging . Despite all of these applications for gold nanoparticles, there is still little knowledge as to how these colloid systems effect microbial environments. Silica nanoparticles are favorable because they are inexpensive, easy to produce, and have surface hydroxide groups that make them easy to functionalize
Results and discussion
Nanoparticles have a tendency to agglomerate in solution due, in part, to the characteristics of the liquid medium with the addition of salt. In regards to the nanoparticles and microbial cell interactions, this will greatly affect the behavior of the cells. Non-agglomerated particles suspended in solution are preferable for testing purposes because of the following:
1) free moving, single unit particles have more contact with microbes.
2) translocation through the cell membrane will be accelerated due to size.
LB media contains a high salt concentration (0.2 M) that may contribute to the agglomeration of the nanoparticles. The surface charge on the nanoparticles in solution allows the nanoparticles to attract to one another because of the influence of ions from the salts, therefore resulting in the formation of large agglomerates [8, 9, 10]. As a result, the nanoparticles may fall out of solution and settle to the bottom of the shake flasks.
Preliminary studies were performed to determine if nanoparticles affect the growth of microbial cells by studying cell cultures in the presence of several inorganic nanoparticles. Experimental evidence indicated that the interactions between E. coli and the nanoparticles used during this study were nonspecific. E. coli showed no overt signs of growth inhibition using the methods presented in this paper. Of course, it is possible that there may be more subtle changes in cell function and behavior detectable at the gene or protein level. For the purpose of this study, it was important to show preliminary results that describe the effects of inorganic nanoparticles under normal growth conditions using known methods for measuring microbial cellular growth. However, as a cautionary note, the results presented are not meant to be generalized beyond the material and biological systems and conditions reported here.
Characteristics of the inorganic nanoparticles used in experimentation.
Mean radius (nm)
Concentration per flask (g/mL)
60 ± 1.3
3.3 × 10-2
80 ± 2.5
2.2 × 10-3
amorphous silica, crystalline iron oxide
30 ± 0.15
1.1 × 10-4
Culture media and culture conditions
For rapid growth of the microbial cells, Luria Bertani (LB) medium was prepared and sterilized for each experiment. A set of 250 mL shake flasks were also sterilized before experimentation. 100 mL of LB medium was transferred to each flask. Various concentrations of nanoparticles were carefully placed into each flask, leaving one as a control to track the normal growth of the microbial cells without nanoparticles. Experiments were performed using both a negative control (flask containing cells plus media) and a positive control (flask containing nanoparticles plus media). Both of the negative and positive control values obtained from optical density measurements were subtracted from the experimental values (flasks containing cells, media, and nanoparticles). The growth curves represent the difference between the controls and the experimental values.
Each flask was then inoculated with 1 mL of E. coli (pBR322 JM105) grown in liquid LB medium. The flasks were shaken at 180 rpm and 37°C in a shaking water bath. Optical density measurements from each flask were taken every thirty minutes to record the growth of the microbes from inoculation through late exponential phase using a spectrophotometer set at 600 nm. The growth rate of microbial cells interacting with the nanoparticles was determined from a plot of the log of the optical density versus time.
Particle morphology using transmission electron microscopy
Transmission electron microscopy (TEM) was used to obtain images of the nanoparticles. Silica/iron oxide samples were prepared for TEM imaging by inserting a TEM grid (copper coated with formvar) into dry powder using tweezers to hold the grid. The sample grid was then lightly tapped to remove any excess particles, and the grid was placed in the TEM for imaging. The silica and gold nanoparticles were in suspension, and samples were prepared by inserting the TEM grid into each liquid sample. The sample grids were then allowed to air dry overnight.
Characterization of nanoparticles by dynamic light scattering
One of the more common methods employed to characterize pharmaceutical colloids is dynamic light scattering (DLS). Analysis of the size distribution of the nanoparticles was performed using a DLS autocorrelation tool known as Photocor®. DLS measurements were taken of the nanoparticles in distilled water and in LB growth media. With this procedure, the difference between the behavior of the nanoparticles in solutions with and without salt was compared.
Nanoparticle/cell interaction studies using TEM
After characterizing the various nanoparticles, experiments were conducted to observe the relationship between the iron oxide composite nanoparticles and E. coli in LB media. Cell/nanoparticle interactions were observed using a Zeiss EM10 CA transmission electron microscope at the University of Maryland Biological Ultrastructure Facility. Samples of E. coli were withdrawn at points during late exponential phase (optical density ~0.6). After collection, they were centrifuged and suspended at room temperature in 0.12 M Millonig's phosphate buffer at pH 7.3 and later with 2% glutaraldehyde. The cell pellets were then washed again with buffer, and then secondary fixed with 1% OsO4. At this point, they were washed with distilled water and then postfixed with 2% uranyl acetate, rinsed in buffer and double distilled water, dehydrated in a series of ethanol and propylene oxide immersions, and embedded in Spurr's resin. A diamond knife was used to section the embedded cells. The sections were post-stained with 2.5% aqueous uranyl acetate and 0.2% aqueous lead citrate.
We would like to acknowledge Dr. Isaac Koh for assisting with some of the DLS data collection, Prof. Sara Majetich for supplying the gold nanoparticles for this study, and Tim Maugel of the University of Maryland Biological Ultrastructure Facility for assisting with TEM preparation. This research was partially supported by NSF-MRSEC seed funding through Grant (DMR-0080008). Additional support was made possible through the Sloan Foundation and the GEM Science and Engineering Consortium.
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