Dr. John Ryan
University of Oxford

Interactions of carbon nanotubes (CNT ) with the cell membranes, double stranded DNA and proteins (commences October 2008)

(a) Recent reports have demonstrated the internalisation of single-walled carbon nanotubes (SWNTs) by different living cells, mainly by endocytosis but possibly also via more physical mechanisms. These studies have yielded contradictory data on the toxic effects of SWNTs and their interactions with cell components such as the cell nucleus. However there has been no systematic study of e.g. which parameters determine whether or nor CNTs penetrate membranes, or of the nature of their interaction with different cell organelles. In this project we are beginning a systematic study of the effect of CNT (both SWNTs and multi-walled carbon nanotubes, MWNTs) on both model lipid membranes and on living prokaryotic and eukaryotic cells using AFM, and environmental SEM (ESEM). The influence of CNT length and diameter, surface chemistry (e.g. by introduction of carboxylic groups by oxidation), functionalisation by coating with phospholipid bilayers, proteins or double-stranded DNA on these interactions will be investigated.

(b) Several studies have shown that single-stranded DNA (ssDNA) can wrap around individual SWNTs. However, the interaction between CNTs and double-stranded DNA (dsDNA) has not been studied. This is an important omission, given the (greater) importance of dsDNA within the cell. In this project, we will characterise how dsDNA interacts with CNT (both SWNTs and MWNTs) of different lengths and diameters. Intercalation of CNT between the double strands or DNA wrapping around a CNT would have specific, and different, effects on the structure of a covalent-close circular DNA. These DNA topology changes will be examined by alterations of DNA mobility in horizontal gel electrophoresis. By AFM we will determine the structure and stability of the dsDNA-CNT complex; again, these experiments will be complemented by simulations.

(c) We will also study the potential use of the dsDNA-CNT complex as a carrier of CNTs into cells. The well-studied Gram-negative prokaryote E.coli will be used as a model to investigate the transport of CNT and dsDNA-CNT through the membrane. A number of different methods that change the membrane permeability, such as electroporation, will be explored.

(d) One of the main objectives of the project will be to control the internalisation of CNTs by the eukaryotic nucleus in order to study their effects on DNA metabolic process such as replication, transcription, and DNA segregation. The interaction of CNTs with genomic DNA and with relevant proteins during the different states of cell cycle will be investigated. dsDNA-CNT and other functionalised CNT will be designed to directly visualise under fluorescence microscopy transport inside the eukaryotic cell. We will use yeast (S. cerevissiae) as a model unicellular eukaryotic organism. Additionally, in order to address the potential role of CNT for drug delivery, we will functionalise CNT with anticancer drugs (e.g. Amsacrine or Epipodophyllotoxins) which act on the eukaryotic enzyme DNA topoisomerase II, an essential protein of the cell cycle which is involved in tumour cell proliferation.