Grantee Research Project Results

During the first year of this project, we focused on creating our first set of nanomaterials, fullerenes with varying functional groups that were positively, negatively, or neutrally charged and had differing structural attachments (covalently or not covalently functionalized). We then used these materials to probe our immune model system. Our investigations have focused on testing a wide concentration range (0.1-100 ug/mL) for each nanomaterial to determine toxicity to macrophage cells using two tests for cytotoxicity: (1) the metabolic reduction of resazurin and (2) lactate dehydrogenase activity (LDH). The goal was to determine if these particles were cytotoxic and at what concentration. In addition, this information was then used as a range finding experiment to determine the concentrations to use for determining the impact on immune function. We wanted a concentration where we would see immune stimulation but not see a complete destruction of the cellular system. We used concentrations of nanomaterials below cytotoxic levels to test for effects on targeted gene expression. We chose TNFα (tumor necrosis factor) and COX2 (cyclooxygenase) as representative of proinflammatory genes; IFNα (interferon) and IP-10 (interferon inducible protein) as genes associated with the antiviral response; and IL10 (interleukin) and TGFβ-1 (transforming growth factor) as representative of the antiinflammatory (alternative/Th2) pathway. Each gene was assayed for expression in several tissue concentrations using RT-PCR (QPCR) to investigate any dose-dependent effects on expression.

 

In the second year of our project, we continued creating nanomaterials with differing functionalization by including single and multi-walled carbon nanomaterials. We narrowed our candidate genes down to ILl and IFN as the best indicators of inflammatory and antiviral responses. These were used to screen all further nanomaterials.

 

In the final years of this project, we focused on a narrow set of concentrations of nanomaterials and those that caused the greatest immune response (single walled nanotubes with varying functionalization) and used custom microarrays to determine if there were unique genomic signatures associated with each nanomaterial exposure. In addition, we examined whether genomic response provided an indication as to whether the nanomaterials were differing in their immune effects based on their structure or functionalization.

 

Our results indicate that fullerenes as well as single wall nanotubes and multiwall nanotubes are stimulatory to primary immune cells and they exhibit a dose-response effect and the immune cells react to each differently. Functionalization of nanomaterial may have an even greater impact on response than the core structure of the nanomaterial.

 

Side-chains used on NPs may have significant effects on their own. For example, cyclodextrin is a stimulator of inflammatory gene expression in trout macrophages. Surfactants used to solubilize NPs may have significant effects on gene expression. Deoxycholate and tetrahydrofuran are both stimulatory of inflammatory gene expression in trout macrophages.

 

Because we found a significant impact of surfactants on stimulation of our cell culture system, we moved completely away from using these suspensions and instead stirred our particles into suspension without the surfactant present with the understanding that this creates a slight alteration of the external aggregation. Of course, the functionalized nanoparticles were created to increase solubility and negate the need for the surfactant in the first place. Because of these properties, they are particularly attractive for industries and therefore more relevant exposures.

 

We determined that immune cell viability was not impacted by nanomaterial exposures up to 10 µg/mL concentrations. However, surfactants as well as chemicals used for functionalization impact cell viability slightly can can cause an inflammatory reeation that can be equal to that of the nanomaterials themselves. We have found that despite a lack of change in cell viability, nanomaterials vary in their stimulation of the immune response such as IL1. All nanomaterials we have tested cause an increase in candidate proinflammatory genes that is equivalent to stimulation of positive controls at the highest concentration of 10µg/mL but as the concentration drops to 1 or .1 µg/mL, there are differences in inflammatory responses with nanomaterials with differing chemistries and in a dose dependent fashion. There are a couple of nanomaterials that appear to be immune suppressive at all exposures tested. This would indicate that the dose that ultimately enters the organism will be extremely important to determine potential immune responses.

We have found that, despite a lack of change in cell viability, nanomaterials vary in their stimulation of the immune response as indicated by QPCR of genes indicative of an inflammatory response such as IL1. All nanomaterials we have tested cause an increase in candidate proinflammatory genes that is equivalent to stimulation of positive controls at the highest concentration of 10 ug/mL, but as the concentration drops to 1or .1 ug/mL, there are differences in inflammatory responses with nanomaterials with differing chemistries and in a dose dependent fashion. There are a couple of nanomaterials that appear to be immune suppressive at all exposures tested. This would indicate that the dose that ultimately enters the organism will be extremely important to determine potential immune responses.

A microarray specifically designed to probe the global immune response of macrophages was used to examine differences in gene expression across nanomaterial treatments and to compare these expression patterns to traditional immune stimulants from viruses and bacteria. Even though each nanomaterial caused a similar inflammatory response in macrophage cells at 5 ug/mL exposures, the global gene expression differed with functionalization of nanomaterial and with core structure. This was mainly in the form of the types of genes expressed in each nanomaterial treatment indicating an overall difference in response to each nanomaterial.