Abstract Background: The use of nanomaterials in biomedical applications is increasing exponentially, with multiple devices being explored for cancer imaging and therapy. For them to enter the clinic, however, it is very important that the toxicity as well as other effects on the tumor microenvironment is assessed accurately over time. In this study, we investigated the cell motility and the cell mitosis of proliferating human endothelial cells, critical components of the tumor microenvironment, while they are treated with PAMAM dendrimer nanoparticles using time-lapsed video microscopy. Conventional in vitro cytotoxicity assays give a cumulative result of the whole cell population whereas the time-lapsed video microscopy allows one to visualize continuously and analyze each cell separately for the cytotoxicity effect of the treatments. We also assess another critical component of the microenvironment, the “irritability” or inflammability of nanoparticles toward human lung macrophages using rocket immuno-electrophoresis, a powerful and quantitative immuno-analytical technique consists of a two-dimensional immunoelectrophoresis with a combination of conventional electrophoretic separation and electro-immunodiffusion. Purpose: Assessment of key tumor microenvironment in the cells exposed to PAMAM dendrimer nanoparticles Experimental: (a). HUVEC were treated with PAMAM generation-5 parent dendrimer nanoparticle and analyzed frame-by-frame using time-lapse video microscopy against the untreated cells. (b) Human lung macrophage cells were treated with PAMAM generation-5 dendrimers of different levels of surface positive charges or positive control turpentine. After 24 hours, the conditioned macrophage medium was added onto rat hepatoma cells. After 24 hours, the medium is removed and analyzed by rocket immunoelectrophoresis for inflammation markers, α1-acid glycoprotein and haptoglobin, two well known acute phase reactants whose levels in the serum increase following inflammatory stimuli. Results and Conclusions: (a) time-lapse video microscopy permitted the quantitative assessment of cell death and mitosis in treated and untreated endothelial cells. (b) In inflammation assays, fully positive surface-charged dendrimers, expected to have the most toxicity, were safe even at high physiologic concentrations (at or below 0.1 μM level) and did not exhibit any detectable toxicity (cell kill) or have any inflammatory effect on human lung macrophages in these concentrations. Neutralization of amino groups made these materials completely non-toxic, even at non-physiologic concentrations (1 μM) and none showed any significant inflammation effect compared to well known irritant turpentine, based on acute phase reactants α1-acid glycoprotein and haptoglobin levels whose levels in the serum increase following inflammatory stimuli. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2262.