| Nanotechnology and nanomaterials are increasingly applied in various aspects of human life, such as energy, environment, electronics and biomedicine. However, people know little about their biological effects. With more and more studies on nanotoxicities have been reported, nanomaterials'safety has caused wide public concern. The key problem is to understand basic biological behavior and mechanisms of nanomaterials. Here, we selected several important types of nanomaterials to study their biological activities. Using multiple physical, chemical and biological approaches, we found important rules on nanoparticles'protein binding, cell uptake and effects on cellular signaling transductions. These findings offer theoretical explanations to nanomaterials biological effects and help elucidate novel interaction and regulation mechanisms. These findings also provide methodological reference to effectively reduce nanotoxicity and improve biocompatibility, and make functional nanomaterials to be probably used as therapeutic agents.We firstly studied nanoparticles'protein binding. The protein binding propensity of nanoparticles determines their in vivo toxicity and their fate to be opsonized and cleared by human defense systems. In this work, protein binding mechanisms of pristine and functionalized multi-walled carbon nanotubes (MWCNT) were investigated by varying MWCNT's diameters, nanotube surface chemistry, and proteins using steady-state and time-resolved fluorescence, and circular dichroism (CD) spectroscopies. The MWCNT with a larger diameter (～40 nm) generally exhibited stronger protein binding compared with those with a smaller diameter (～10 nm), demonstrating that the curvature of nanoparticles plays a key role in determining the protein binding affinity. Negative charges or steric properties on MWCNT enhanced binding for some proteins, but not others, indicating that the electrostatic and stereochemical nature of both nanotubes and proteins govern nanotube/protein binding. Protein fluorescence lifetime was not altered by the binding while the intensity was quenched indicating a static quenching through complex formation. The binding-induced conformational changes were further confirmed by CD studies.Effects on cellular signaling pathways can occur when nanotubes interact with cell surface receptors or with intracellular proteins. The evaluation of cell uptake and intracellular location of SWCNT-COOH are crucial for understanding its biological impact mechanism. CNT can readily penetrate various biological barriers in mammals, plants, and microorganisms. However, the mechanism of cell uptake and cellular transfer of CNT is not fully understood. The current explanations on cell uptake of CNT, their intracellular translocation, and subcellular localization are still controversial. To fill this gap, we examined cell uptake of surface charged MWCNT using TEM. We observed direct membrance penetration, endocytosis, endosomal leakage and nuclear translocation of MWCNT. Previous reports and our own experimental results are consistent with a working model for the cell uptake of CNT. CNT clusters are taken up by cells through energy-dependent endocytosis process. The CNT bundles become unpacked in the endosomes and generate single nanotubes that escape endosomes by penetrating endosome membrane and entering the cytoplasm. Alternatively, the highly dispersed single CNT cross cell membrane and enter cells directly by penetrating cellular membranes. All CNT are finally recruited into lysosomes for excretion. The model will have major impacts on both drug delivery and toxicity studies of CNT. For example, all cellular CNT may be exposed to cytoplasm so that the unexpected interactions with cellular functional molecules are likely to happen.After nanoparticles enter cells, they will inevitably interact with cellular components and time-and dose-dependent cellular responses are likely to occur. We use a novel real-time cell-based electronic sensing (RT-CES) technology to dynamically monitor cellular responses to carbon nanotubes. This approach is based on the parallel impedance measurement of attached cells using electronic sensors integrated in wells of 96-well E-plate. It measures the real-time multi-parameter index of cell growth named cell index (CI), which reflects the cell proliferation, morphology, attachment and spreading. The label-free, real-time and high-throughput assay overcomes many drawbacks in current optical based cytotoxicity assays in carbon nanotubes research, and enables dynamic monitoring of cellular responses to carbon nanotubes. Using this assay, we obtained dynamic cellular response curves, time-dependent IC50s and cell growth slopes which can not be obtained by conventional assays.Since nanoparticles intrinsically interact with proteins and enter cells and cause dynamic cellular responses, it's urgent to elucidate the underlying molecular mechanisms of a nanoparticle's cellular behavior, including what signaling pathway is affected, what genes are changed and what are the consequences. Through our dynamic monitoring of cellular responses and evaluation of genome expression, as well as other cellular biological approaches, we discovered that SWCNT-COOH inhibited cell proliferation via a non-apoptotic mechanism, which is different from effects caused by pristine CNT. On the basis of SWCNT-COOH's perturbations on cells, expression of genes and protein, and protein phosphorylations, we conclude that SWCNT-COOH suppresses Smad-dependent bone morphogenetic protein (BMP) signaling pathway and down-regulates Id proteins. These molecular actions cause cell cycle arrest at G1/S transition and inhibit cell proliferation. The specific suppression of BMP signaling and Id proteins by SWCNT-COOH demonstrates non-apoptotic effects of functionalized CNT on human cells. This finding may have potential therapeutic value to treat human diseases related to Id proteins or BMP signaling such as breast cancer and bone diseases.Although nanoparticle/protein binding and the cytotoxicity of nanoparticles have been separately reported, there has been no study linking the nature of nanoparticle/protein clusters to cell uptake and the dynamic cellular responses. We report here that water soluble iron oxide based magnetic nanoparticles (MNPs) with different sizes and surface chemistry bind different serum proteins in terms of protein identity and quantity without changing the protein secondary structures. Carboxylated MNPs resulted in higher cytotoxicity and PEG-coating reduced both cell uptake and the cytotoxicity. Smaller MNPs (especially the carboxylated one) bind more serum proteins, are much less taken up by cells compared to larger particles, yet elicit more dynamic cytotoxic responses. Besides the intrinsic effects of size and surface charge of the water soluble MNPs, the cellular effects of MNPs/protein clusters were also attributed to the identity and quantity of the adsorbed proteins rather than the binding-induced new epitopes on the proteins.The rapid development of nanotechnology, especially the increasingly emerging nanostructures requires prerequisite bio-activity evaluations. Core/shell iron/carbon nanoparticles (Fe@CNPs) are novel nanomaterials which have potential applications in magnetic resonance imaging (MRI), magnetic hyperthermia and drug delivery, etc. However, their interactions with biological systems are totally unknown at present. To elucidate their potential cytotoxicity and explore the relationship of biocompatibility with their surface chemistry, we synthesized different types of polymer grafted Fe@CNPs and studied their dynamic cellular responses, cell uptake, oxidative stress and their effects on cell apoptosis and cell cycle. The results show that cellular biocompatibility of Fe@CNPs is both surface chemistry dependent and cell type specific and generally non-toxic except for the carboxyl modified Fe@CNPs. Our study indicates that these novel materials can be used for further functionalizations and widely applied in many fields.
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