Differential Effects Of Multiwalled Carbon Nanotubes And Fullerene C60 On In Vitro Astroglial Cells

Nanomaterials refer to materials that have at least one dimension 100 nanometres or less. Materials at nanoscale not only possess extraordinary physic-chemical properties not seen at macroscale but also can have unique biological effects which are foundamentally determined by their physic-chemistry. A best pair of examples is multiwalled carbon nanotubes (MWCNTs) and flullerene C60 (C60) which are carbon allotropes of distinct nano structures. Neuroscience is a field where the interactions of theses two types of carbon macromolecules with neverous system are a focus of research attention and where the biological properties and application potential of MCNTs and C60 are most extensively explored. Astrocytes, or astroglial cells, are a dominant and highly active component of the central nervous system (CNS) that play pivotal structural and functional roles and outnumber neurons by about 10 fold in the human brain. The purpose of the present project was to investigate the interactions of MWCNTs and C60 with astrocytes and transformed astrocytes i.e. glioma cells and to compare the dfferent biological effects of MWCNTs and C60.Monitoring of cell viability is a regular but essential part of work in any cell-based study. Two widely used assays i.e. MTT reduction and resazurin reduction were first performed to evaluate the effects of MWCNTs and C60 on the viability of in vitro rat astrocytes, but yielded contradictory results regarding MWCNTs, indiating unsuitability of these methods for assaying cellular responses to MWCNTs. To provide accurate information on MWCNTs' cellular effects, a series of flow cytometry-based procedures were then developed, validated and effectively applied. C60 was also tested in comparison with MWCNTs. Briefly, cells exposed to MWCNTs were stained by a panel of fluorescent probes for different aspects of cell function, including 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1),3,3'-Dihexyloxacarbocyanine, iodide (DiOC6(3)), rhodamine 123 (R123) and fluorescein diacetate (FDA). Probe indications were characterized and probe staining performed both in an "in situ" and "ex situ" manner on MWCNTs-or C60-exposed cells were analyzed by flow cytometry. JC-1 and DiOC6 (3) staining were found good indicators of mitochondrial membrane potential (ATm). WhileΔ(?)m contributed to R123 uptake, intracellular accumulation of R123 was largely determined by the function of P-glycoprotein, a cell membrane bound efflux protein. Multidrug-resistance associated protein, another efflux protein, was found to determine cellular FDA staining. Decreased JC-1 and DiOC6(3) uptake were observed in MWCNTs-exposed cells but couldn't be attributed toΔ(?)m disruption. In contrast, cellular staining of R123 and FDA was enhanced after MWCNT exposure. Mode of dye loading was found to significantly affect the outcome of cellular dye staining after MWCNT exposure. Compared with MWCNTs, C60 generally exerted insignificant influence on the staining of all probes. In summary, fluorescent probe staining in combination with flow cytometry, after careful validation, can provide good assay of cellular reposnses to the exposure of MWCNTs and C60. Astrocyte viability appeared to be maintained in the presence of MWCNTs and C60 at concentrations and for the duration tested. However, certain aspect of cell function relating to the cell membrane might be affected.One of the many functions of the cell membrane is to execute substance transport into and out of the cell and cell membrane transporter proteins like P-glycoprotein (Pgp) and multidrug resistance related protein (MRP) are a key mechanism therefor. As observed in my work presented in Part 1, rat astrocytes exposed to MWCNTs but not C60 displayed increased contents of rhodamine 123 (R123) and fluorescein (Flu) which are respective substrates of Pgp and MRP. It was thus postulated that MWCNTs might be able to affect cross-membrane drug transport, particularly that mediated by Pgp and MRP. To test this hypothesis, we investigated the influence of MWCNTs, and C60 as a comparison, on the transport of several compounds across the cell membrane of rat astrocytes using flow cytometry. These compounds were fluorescein diacetate, carboxyfluorescein diacetate, rhodamine 123 and doxorubicin which are prosubstrate/substrates of multidrug transporter proteins. Results showed that MWCNTs significantly inhibited cellular uptake of doxorubicin but not the other drugs and mode of loading made a significant difference in doxorubicin uptake. Retention of fluorescein, carboxyfluorescein and rhodamine 123 was remarkably higher in MWCNTs-exposed cells after an efflux period. Kinetics study also demonstrated slowed efflux of intracellular fluorescein and rhodamine 123. C60 generally had no siginificant effect on the uptake and efflux of the tested substrates. Data presented in this paper suggest that MWCNTs could affect drug transport across cell membrane.MRP-mediated drug transport in a human glioma cell model In the study on the interactions of carbon nanotubes with living cells, the cell membrane deserves particular attention as it provides the first interface to initiate CNTs-cell interactions. In my work presented in Part 2, MWCNTs were demonstrated to be able to affect cross-membrane drug transport, especially that mediated by Pgp and MRP in a rat astrocyte model. In the present work, the inhibiting effect of MWCNTs on the MRP mediated fluorescein efflux in a human glioma cell model was also demonstrated. To provide clues to explanation of this effect, intracellular glutathione content and reactive oxygen species production were determined as fluorescein is a specific substrate of cell membrane MRP whose transport activity requires glutathione which can be depleted under oxidative stress. The plasma membrane potential was also probed as the susceptibility of fluorescein efflux to modulation of the plasma membrane potential has been documented. Results showed a remarkable decrease in cellular glutathione level as well as an increase in reactive oxygen species production. Probe staining also indicated decreased plasma membrane potential. The data suggested that multiwalled carbon nanotubes may affect the transport activity of cell membrane multidrug resistance-related protein through reduction of intracellular glutathione content. Hypopolarization of the plasma membrane may also contribute to MWCNTs'effect. In vitro rat astrocytes and human glioma cells were studied for their interactions with prestine MWCNTs and C60. Principle and original discoveries of my work are 1) Investigation methods and approaches commonly used for conventional drug studies might yield misleading results when used for biological study of nanomaterials like MWCNTs. Optimized and validated flow cytometry based assays are convenient and effective for studying cellular effects of nanomaterials like MWCNTs. Yet, caution and discretion are still needed to ward off false judgements.2) Prestine MWCNTs and C60 both have little influence on cell viability, but drug transport across cell membrane, particularly that mediated by transporter proteins can be affected by MWCNTs but not C60. MWCNTs and C60 are both carbon macromolecules. But they can have distinct biological effects as demonstrated in the present work, probably due to their different nanostructures.3) The mechanisms of MWCNTs'effects may involve cell membrane perturbation and depletion of intracellular glutathione due to oxidatice stress.