Polymeric nanoparticles have been investigated for a wide range of potential applications, especially in the biomedical field in terms of drug, gene, and vaccine delivery vectors as well as for MRI contrast agents for diagnosis. Given the potential widespread use of both the series of polymeric nanomaterials (PAMAM dendrimers, PNIPAM and NIPAM/BAM copolymer nanoparticles), there is an increasing need for information regarding the human health and environmental implications of exposure to these polymeric nanomaterials. In terms of human exposure, considerable attention has been devoted of late to the potential effects of exposure to Nanomaterials. However, the field of eco-(nano) toxicology is still relatively new and there is a dearth of quantitative structure activity relationships established for nanomaterials. Thus, this study has utilised both human and aquatic models. In the case of polymeric nanoparticles, the structurally well defined and variable macromolecules can also provide a further basis upon which to establish structure activity relationships governing eco and mammalian-toxicological responses which may serve to develop a fundamental understanding of their interactions and as guidelines for the future prediction of responses. The systematically varied molecular PAMAM dendrimer nanostructures potentially provide a route towards an understanding of the dependence of the interactions on the physico-chemical properties of nanomaterials. In case of PNIPAM and NIPAM/BAM nanoparticles, a systematically varied surface morphology is achieved as the ratio of BAM increases, the amount of N-H groups exposed at the surface decreases, and the amount of -C-(CH3)3 groups increases, reducing the hydrophillicity of the resulting copolymer. Both the polymer particles were selected to understand the correlation between physico-chemical properties and toxicological impact of these nanomaterials to the human health as well as to the environment.
PNIPAM and NIPAM/BAM nanoparticles are well known thermoresponsive particles and to the best of our knowledge there is no eco and mammalian toxicity data of PNIPAM and NIPAM/BAM co-polymer nanoparticles available to date. The most sensitive ecotoxicological assay for PNIPAM and NIPAM/BAM 85:15 nanoparticles was the immobilisation of Daphnia magna (48 hour EC50) and for NIPAM/BAM 65:35 and NIPAM/BAM 50:50 nanoparticles was the Microtox® assay (Vibrio fischeri, 5 minutes EC50). The least sensitive bioassay was Pseudokirchneriella subcapitata (72 h EC50) for the four nanomaterials tested. An important conclusion from the study therefore is that the sensitivity of each assay is dependent on the physico-chemical characteristics of the particle, emphasising the importance of a multi-trophic approach. As the ratio of BAM increases in the copolymer nanoparticles the toxicity was increased in all the test species, despite the fact that the particles with the highest ratio of BAM were highly aggregated. The toxicity trend for different nanoparticles was PNIPAM  NIPAM/BAM 85:15  NIPAM/BAM 65:35  NIPAM/BAM 50:50, which suggests that there is a significant effect due to particle hydrophobicity and the surface free energy (Lynch et al., 2005). This is confirmed by the correlation of the toxic response with the observed zeta potential of the particles in the medium. The correlation of the toxic response in Daphnia magna with the reduction in zeta potential points towards a contribution of secondary effects due to modification of the medium. No dependence of the toxic response on the particle size was observed however. Nevertheless the study gives a clear dependence of the toxic response on the particle composition pointing towards structure-activity relationships.
Mammalian toxicological evaluation of PNIPAM nanoparticles indicated no significant cytotoxic response in HaCaT and SW480 cells, and that these particles are biocompatible in nature. No significant difference in the cell viability upon exposure of either cell type to PNIPAM nanoparticles was found after 24h, 48h, 72h and 96h of exposure at concentrations ranging from 12.5 to 1000 mg/l. The biocompatibility of the PNIPAM nanoparticles is further confirmed by the genotoxicity results, as there is no significant difference in the % tail DNA and olive tail moment (OTM) in either the HaCaT and SW480 cells upon exposure of the particles. Fluorescently labelled PNIPAM particles are clearly seen to be internalised by HaCaT and SW480 cells after 24hrs, and are specifically localised in lysosomes.
The observed interaction of the PNIPAM nanoparticles with the two different mammalian cell lines and the interpretation of the consequences of the particle fate and behaviour within the cells is an indication of the biocompatibility of these polymer particles. In addition to this, it was observed that PNIPAM nanoparticles do not elicit a ecotoxicological response, hence it is also an eco-friendly polymer particle. The data presented here would suggest that these particles have significant potential as drug delivery agents in the form of hydrogels or as scaffolds in the field of tissue engineering.
However the ecotoxicological study of Polyamidoamine (PAMAM) dendrimers demonstrated significant eco and cytotoxicological responses at concentration ranges from 0.13 ïM to 16.30 ïM. For all generations of PAMAM dendrimer tested, the Daphnia magna was shown to be the most sensitive test model, the RTG-2 cell line being the least sensitive. The ecotoxicological response was seen to correlate well with the generation of PAMAM dendrimers and therefore with the particle surface area. The surface chemistry is unaltered in successive generations, and thus a clear and direct relationship between the physical parameter and the toxic response is inferred. The physico- chemical characteristics, most notably the zeta potential of the particles, were seen to change dependant on the dispersion medium, however, and the correlation of the toxic response to these changes may point towards an interaction with the medium resulting in a change in effective composition as an underlying source of the toxic response. Successive generations present a larger number of surface amino groups for interaction with the media, and thus a larger toxic response. Such an indirect effect can not be considered as the sole origin; however, as is seen by comparison of the PAMAM dendrimers with the NIPAM/BAM copolymer nanoparticles and mechanisms of internalisation resulting in a direct toxic response should be investigated for all models.
A significant genotoxicity and apoptosis response in PLHC-1 cells was observed upon the exposure to PAMAM dendrimers. The generation dependence (G6  G5  G4) of the production of increased intracellular ROS, DNA damage, apoptosis and the cytotoxicity in the PLHC-1 cells, indicates the direct effects of the positively charged surface amino groups.
The immunotoxicity of PAMAM dendrimers was investigated in mouse macrophage cells (J774A.1) in vitro at a concentration of 0.013 to 6 ïM. Generation dependent immunotoxicological response of PAMAM dendrimer was observed in J774A.1 cells. The generation dependence (G6  G5  G4) of the production of increased intracellular ROS, inflammatory mediators and the cytotoxicity. 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.
The ecotoxicological study of the NIPAM/BAM series of nanoparticles shows significant toxic effects at higher concentration. As the ratio of BAM increases in the nanomaterial composite there is a systematic increase of toxic response. However, in case of PNIPAM nanoparticles, no toxicological response observed with mammalian cells even at higher doses.
PAMAM dendrimers shows significant toxic response at the lower concentrations to both the fresh water ecological organisms and the mammalian cells. Clear structure property relationships are indicated for the toxic responses in both cases.
An evolving paradigm of toxic responses to nanomaterials begins with the generation of intracellular ROS, followed by lysosomal and mitochondrial damage, which leads to DNA damage, mutation, apoptosis and finally cell death. PAMAM dendrimer induced cell death has been demonstrated to follow this mechanistic pathway.
These Nanomaterials, having systematic structural variations in molecular weight, surface primary amino group, and size, represent ideal model systems to explore structure property relationships governing toxicological response. In addition, PAMAM dendrimers hold potential as gene transfecting agents due to the positive charge on the surface and are also employed as vaccine delivering agents Understanding their mode of interaction and cellular transport can lead to improved guidelines for the design of drug delivery systems.
Nanoparticles elicit a wide range of intracellular responses depending on their physicochemical properties, intracellular concentrations, and duration of exposure, subcellular distributions and interactions with biological molecules. Endocytic pathways include pinocytosis, the formation of caveolae and clathrin, and caveolae/
Clathrinin dependent uptake. Although the cell types and their states of differentiation can determine the choice of pathway, the physicochemical properties and surface reactivities of nanoparticles are also important.
According to a recent hypothesis by Nel et al., 2009, cationic nanomaterials induce cell death due to the proton sponge effect leading to endosomal/lysosomal damage and the induction of cytotoxicity. For example PEI coated particles bind with high affinity to lipid groups on the surface membrane and are endocytosed in the tight-fitting vesicles. Once these cationic nanoparticles enter into an acidifying lysosomal compartment, the unsaturated amino groups are capable of sequestering protons that are supplied by the v-ATPase (proton pump). This process keeps the pump functioning and leads to the retention of one Cl- ion and one water molecule per proton. Subsequent lysosomal swelling and rupture leads to particle deposition in the cytoplasm and the spillage of the lysosomal content (Nel et al., 2010).
In addition to entry into endosomal compartments, engineered nanoparticles exert important effects on other organelles (Xia et al., 2006 and 2008). Particularly noteworthy is the impact on lysosomal function, which is key to nanoparticle use as a delivery device, as well as for nanomaterial toxicity. The first relates to the potential of aminolabelled polystyrene, cationic dendrimers and polymers (for example PEI) complexed to DNA (polyplexes) to enter the lysosomal compartment. The lysosomal proton pump, which is responsible for acidification, is key to understanding the proton sponge hypothesis, which posits that unsaturated amines on the material surface are capable of sequestering protons (Xia et al., 2008), keeping the pump going and leading to the retention of one Cl- anion and one water molecule for each proton that enters the lysosome. Ultimately, this process causes lysosomal swelling and rupture, leading to particle deposition in the cytoplasm. This process includes triggering of the Ca2+regulated permeability transition pore in mitochondria (Xia et al., 2008), potentially leading to decreased ATP production and cellular apoptosis. Spillage of lysosomal enzymes can also lead to the activation of Bid and Bax proteins and procaspases (Xia et al., 2008).
These are the process happening with cationic Nanomaterials and these pathways may be core to the action of the cationic amino terminated PAMAM dendrimers. Therefore, in future studies, it is important to explore and to understand the complete pathways leading to cell death.
