Functional and Pathological Analysis of Biological Systems Using Vibrational Spectroscopy with Chemometric and Heuristic Approaches

Vibrational spectroscopy (Raman and FTIR microspectroscopy) is an attractive modality for the analysis of biological samples since it provides a complete non-invasive acquisition of the biochemical fingerprint of the sample. Studies in our laboratory have applied vibrational spectroscopy to the analysis of biological function in response to external agents (chemotherapeutic drugs, ionising radiation, nanoparticles), together with studies of the pathology of tissue (skin and cervix) in health and disease. Genetic algorithms have been used to optimize spectral treatments in tandem with the analysis of the data (using generalized regression neural networks (GRNN), artificial neural networks (ANN), partial least squares modelling (PlS) and support vector machines (SVM), to optimize the complete analytical scheme and maximise the predictive capacity of the spectroscopic data. In addition we utilise variable selection techniques to focus on spectral features that provide maximal classification or regress ion of the spectroscopic data against analytical targets. This approach has yielded interesting insights into the variation of biochemical features of the biological system with its state, and has provided the means to develop further the analytical and predictive capabilities of vibrational spectroscopy in biological analysis

Eco and In Vitro Mammalian Toxicological Assessment of Polymeric Nanomaterials

Eco and in vitro mammalian toxicological assessment of N-isopropylacrylamide (NIPAM) / N-tert-butylacrylamide (BAM) copolymer nanoparticles and Polyamidoamine (PAMAM) dendrimers were performed in a range of test models. The particle sizes of the copolymer nanoparticles and PAMAM dendrimers (G4, G5 and G6) were measured in Milli-Q water, Algae Media, Daphnia Media, Microtox Diluent and cell culture media. The zeta potential of PNIPAM (Poly- N-isopropylacrylamide) and NIPAM/BAM copolymer nanoparticles measured in the different media was seen to correlate well with the ratio of BAM monomer and therefore the hydrophobicity of the particles. Ecotoxicological studies of the NIPAM/BAM copolymer nanoparticles and PAMAM dendrimers was performed using four test species; Vibrio fischeri, Pseudokirchneriella subcapitata, Daphnia magna, Thamnocephalus platyurus. The cytotoxicity of PNIPAM, NIPAM/BAM 85:15 copolymer nanoparticles and PAMAM dendrimers (G4, G5 and G6) was evaluated in RTG-2 (rainbow trout gonadal cells) and PLHC-1 (hepatocellular carcinoma in an adult female topminnow (Poeciliopsis lucida) cells. The generation of intracellular reactive oxygen species, genotoxicity and apoptosis was evaluated upon the exposure of PLHC-1 cells to PAMAM dendrimers. The mammalian cyto- and geno- toxicity of PNIPAM nanoparticles were analysed in HaCaT (an immortal non-cancerous human keratinocyte cell line) and SW 480 (a primary adenocarcinoma cell line of the colon) cells. The immunotoxicity of PAMAM dendrimers was evaluated in mouse macrophages (J774A.1 cells). Inflammatory markers like IL-6 (Interleukin-6), TNF-a (Tumour necrosis factor-a) and MIP-2 (Macrophage inflammatory protein-2) expression were measured by ELISA (Enzyme linked immuno sorbent assay). In the case of the NIPAM/BAM series of nanoparticles, Abstract ii the ecotoxicological response was seen to vary systematically with the ratio of BAM monomer and therefore with the zeta potential of the nanoparticles. The toxic response in Daphnia magna was seen to also vary systematically with the reduction in zeta potential pointing towards a contribution of secondary effects due to modification of the medium. PNIPAM nanoparticles show excellent biocompatibility in HaCaT (immortalised non-cancerous human keratinocyte) and SW480 (primary adenocarcinoma of colon) cells, as no significant cyto or genotoxicity response has been observed even at high dose, although the particles were internalised by the cells within 24h. In the case of PAMAM dendrimers, a significant eco and cytotoxicological response was recorded at particle concentrations from 0.129 μM (7.4 mg l-1) to 16.55 μM (235.1 mg l-1) and Daphnia magna was found to be the most sensitive test species, the RTG-2 fish cell line the least sensitive. Consistent with the results of the cytotoxicity assays, a generation dependent intracellular ROS, DNA damage and apoptosis was observed in PLHC-1 cells upon exposure to PAMAM dendrimers. The immunotoxicity of PAMAM dendrimers was investigated in mouse macrophage cells (J774A.1) in vitro within a concentration range of 0.013 to 6 μM. A generation dependent immunotoxicological response of PAMAM dendrimer was observed (G6 \u3e G5 \u3e G4). A similar generation dependence of the increased production of intracellular ROS and inflammatory mediators was observed. The toxicological response of PAMAM dendrimers varied systematically with the dendrimer generation and therefore with the number of surface amino groups per particle. The mechanism of the toxic response is proposed to be one of localisation of the cationic particles in the mitochondria, leading to significant increase in ROS generation, induction of cytokines production, DNA damage, apoptosis and ultimately cell death. For the cell lines, although spectroscopic studies indicated an Abstract iii interaction with the serum supplement, trends for this interaction do not correlate to those observed for the toxic response. The clear and systematic variations of the observed toxic response with the measured physico-chemical properties point towards underlying structure activity relationships