Effects Of Multiwalled Carbon Nanotubes On In Vitro Multidrug Resistant Glioma Cells

Carbon nanotubes (CNTs) are a kind of carbon allotrope of unique nanostructure, physi-chemical and biological properties. The interactions of CNTs with human body and their potential applications in biomedicine is an area of great interest. Current evidence suggests that CNTs have excellent biocompatibility with the central nervous system (CNS) and CNTs demonstrate enormous promise in diagnostic and therapeutic uses for CNS diseases. Glioma is the most common malignant cancerous disease in the CNS which is often refractory to chemotherapy. Multidrug resistance (MDR) due to over-expression of cell membrane efflux proteins like P-glycoprotein (Pgp) and multidrug resistance related protein (MRP) is a primary cause for the failure of chemotherapy. In the current study we studied the interactions of multi-walled carbon nanotubes (MWCNTs) with in vitro human MDR glioma cells. A series of flow cytometry (FCM) based methods was first established, optimized and validated to investigate MWCNTs'effect on cell viability. Secondly but most importantly, the inhibiting effects of MWCNTs on cell membrane Pgp and MRP mediated transport were demonstrated. The possible mechanisms underlying MWCNTs'effect on MRP mediated transport was also explored. This study includes the following 3 parts:Part 1. Effect of multi-walled carbon nanotubes on cell viability【Objective】To establish, validate and optimize a series of methods based on fluorescent probe staining in combination with FCM to investigate the effect of MWCNTs on different aspects of cell viability.【Methods】The cells were exposed to a gradient of MWCNTs for 24 h and subsequently stained with a panel of fluorescent probes including JC-1,R123,FDA and PI. Staining was performed both in an in situ and an ex situ manner and was analyzed by FCM. Cellular fluorescence was also observed by microscopy and the internalization of MWCNTs observed by transmission electron microscopy. Prior to MWCNTs exposure, the indication of each probe was validated.【Results】Cellular staining of R123 and JC-1 was found to be primarily determined by mitochondrial membrane potential and FDA staining depended on the activity of intracellular unspecific esterase as well as cell membrane integrity. However, there were other factors in the cell that could fundamentally affect the staining of R123 and FDA. In MWCNT-exposed cells, R123 staining was significantly enhanced under in situ condition but varied little from control under ex situ condition. Significant decrease of JC-1 staining was observed in situ cells but not ex situ cells. FDA staining displayed a somewhat bell-shaped pattern in in situ cells but showed minor enhancement in ex situ cells. The number of cells positive of PI staining only slightly increased under both conditions.【Conclusions】Fluorescent probe staining in combination with FCM, after validation and optimization, is a convenient and effective approach for investigating cellular responses to MWCNTs exposure. Within the current experimental framework, human MDR glioma cell viability was generally maintained in the presence of MWCNTs. But certain cellular functions associated with cell membrane were probably affected.Part 2. Effect of MWCNTs on cross-cell membrane drug transport【Objective】To investigate the effect of MWCNTs on substance transport across the cell membrane.【Methods】The cells were respectively loaded with Pgp substrates (R123, DOX) and MRP pre-substrates (FDA, CFDA) following 24 h of MWCNT exposure. Intracellular substrate content was measured by FCM both immediately after loading and after a 60-min efflux period, for evaluation of substrate uptake and retention. Efflux kinetics of intracellular R123 and fluorescein (derived from FDA hydrolysis) was also examined.【Results】MWCNT-exposed cells displayed a remarkable increase in R123 uptake but in contrast a pronounced decrease in DOX uptake. Retention of all substrates was significantly higher in MWCNT-exposed cells, with a commensurate decrease in substrate efflux. Kinetics study also showed slower efflux of intracellular R123 and fluorescein by MWCNT-exposed cells.【Conclusion】MWCNTs could inhibit substance transport across the cell membrane, especially that mediated by Pgp and MRP.Part 3. Mechanisms involved in MWCNTs'effect on MRP transport【Objective】To investigate the possible mechanisms of MWCNTs'effect on MRP mediated transport.【Methods】The effects of modulation of the cell membrane potential (PMP) and intracellular glutathione (GSH) content on MRP mediated efflux of fluorescein was characterized using tool drugs. The PMP and intracellular GSH content were subsequently measured following MWNCT exposure.【Results】MRP mediated efflux of fluorescein was validated to be effectively modulated by the PMP and intracellular content of GSH. Decreased PMP and decreased GSH level with concomitant increased production of reaction oxygen species (ROS) were observed in MWCNT-exposed cells.[Conclusion] Alteration of PMP and depletion of intracellular GSH associated with oxidative stress may be involved in MWCNTs'effect on MRP mediated transport.