The production and use of multi-walled carbon nanotubes (MWNTs) are rapidly increasing world-wide, despite the possible adverse effects they may have on human health. Of particular concern are reports that MWNTs can cause pulmonary fibrosis in lab animals that may lead to mesothelioma, similar to asbestos. The literature is conflicted regarding what physical features of MWNTs cause phagocytic cells to release proinflammatory cytokines that precede pulmonary fibrosis. It has also been suggested that phagolysosome membrane damage caused by MWNTs is a key event leading to cytokine release by phagocytic cells, but what MWNT properties cause damage is also not clear. The broad objectives of the research proposed here are to better understand whether MWNT length and extent of dispersion are factors leading to cytokine release by phagocytic cells and to explore a relatively new approach, laser scanning confocal Raman microscopy, to assess whether MWNTs damage the phagolysosomal membrane en route to eliciting cytokine release. There are two specific aims to achieve the objectives of the proposal: Specific aim 1: To quantify the amount of MWNTs taken up by two types of phagocytic cells and to correlate the uptake with MWNT length, dispersant, agglomeration, toxicity, and the release of proinflammatory cytokines. Two different MWNT types will be studied, long and short. For each type, two dispersions will be prepared with the dispersants bovine serum albumin or Pluronic(R) F-108, highly dispersed and agglomerated. The experimental design will use a new method to measure the actual amount of the MWNTs that accumulate inside cultured phagocytic cells, followed by measuring the release of two cytokines, TNF- and IL-1. The results of this aim will, for the first time, correlate the actual amount of MWNTs inside phagocytic cells with MWNT length, type of dispersant, agglomeration, toxicity, and the release of proinflammatory cytokines. Specific aim 2: To determine the subcellular distribution of MWNTs and potential phagolysosome damage by laser scanning confocal Raman microscopy (LSCRM). LSCRM, a relatively new approach to cell imaging, can directly locate MWNTs inside cells by their unique Raman signature and be used to reconstruct 3D models from confocal sections showing the subcellular distribution of MWNTs. LSCRM can also image membranes in cells based on the C-H bond Raman scattering of membrane lipids. With this combination of capabilities it may be possible to determine whether MWNTs taken up by cells damage the phagolysosome membrane and redistribute throughout the cytosol. Results from this aim should help address whether MWNT length and state of dispersion are factors in damaging the phagolysosome membrane.