| In the past decades, the investigations on nanotechnology have a quick development. Nowadays, there are around60scientific journals in the fields of nanoscience and nanotechnology, most of which are enjoying the climbing impact factors. The development of nanotechnology depends heavily on developing nanomaterials with beneficial properties as well as the optimizations of these properties by physical and chemical ways. Carbon nanotubes (CNTs) are hollow carbon cylinders made of one or more graphene layers and their excellent physicochemical properties have drawn much attention on their potential applications in multiple fields. In biomedicine, studies have shown that, as a drug delivery carrier, CNTs exhibit a high carrying capability, were able to release drug in controllable ways, and are easy to subject to multifunctionalizations. Furthermore, CNTs are also a good imaging contrast agent with a low noise background.Their unique capability to absorb near infrared (NIR) radiation energy can cause local heating, which can be used to lead to tumor cell destruction. Although nanoparticles are not new to human (there are many of them around us in the nature), our understandings on their biological nature are very limit, which has stimulated concerns on their potential toxicity. Investigations have alerted us that CNTs can induce intracellular oxidative stress, inflammation, cell death, malignant cell transformation and lead to asbestos-like toxicity.Interactions with cells cause favorable application potentials as well as nanotoxicities. On the levels of organs and cells, human has gained some knowledge in biological properties of CNTs. For example, we have gained some insight into the bio-distribution in various organs after they enter circulatory system of mouse via different entries (oral administration, intravenous administration, transdermal absorption, and respiratory exposure) and their sub-cellular localization in cells. In comparison, the interaction of CNTs with biological molecules has been relatively scarsely investigated. In this field, CNTs-proteininteractions are mostly studied, which has helped to pave the way to understand mechanically CNTs'biological nature. However, these studies cannot reflect the truth in live cells when CNTs interact with cells. For example, the steric conformations needed for membrane receptor proteins to function normally depend largely on the supporting of cell membrane. Detaching from cell membrane will change their steric conformations and may ruin their biological functions. So it is of practical significance to study the interactions between nanomaterials with biological molecules in the live cells.In the first part of this dissertation (Chapter2), we developed three methods to study the interaction ways between MWCNTs (as well as other nanomaterials) with cell membrane proteins. Some membrane proteins are involved in the regulation of intracellular signaling pathways, which have been reported to be subject to the perturbationof nanomaterials. Nanomaterials bind on cell membrane or enter cells when they encounter cells, so it is reasonable to have the hypothesis that nanomaterials disturb cell signaling by upsetting the functions of membrane receptors or intracellular signaling steps. Even though some researchers found the intracellular signaling steps are disturbed by nanomaterials, whether nanomaterials will interact with membrane receptors keeps unknown. This is partly due to the lack of feasible methods for conducting such studies. Considering CNTs can be regarded as large biological molecules, we try to graft some methods which have been used to study the interactions between biological moleculesto investigate the interactions between CNTs and membrane receptors. These methods include in situ proximity ligation assay (in situ PLA), fluorescence resonance energy transfer (FRET) as well as immunogold with transmission electron microscopy. Based on the successful constructions of MWCNTs-membrane receptor interaction model, we demonstrated that all these methods can be applied for this purpose. These methods will play roles ininvestigating molecular mechanisms ofnanomaterials' biological properties and the relationship between surface modifications with biological effects of nanomaterials.We and other groups have found that both SWCNTs and MWCNTs inhibit cellular bone morphology proteins (BMP) signaling pathway. BMP signaling pathway is a multi-step process and involved in the regulations of a large amount of critical cell functions, therefore, this finding is clinically signalificant. But unfortunately, the exact targeted step by CNTs keeps elusive. In the second part of this dissertation (Chapter3), we used the above methods to investigate the molecular mechanisms underlying the interaction betweensMWCNTs and BMP signaling. Our results indicated that the inhibition on the kinase activity of type2BMP receptor to phosphorylate type1BMP receptor lead to the suppression of BMP signaling activity. This discovery is important in that, it showed that the nanoparticles'effects on cell signaling orginated from their interaction with cell membrane receptor, and the nature of this interaction is nanoparticle-protein interaction. Based on this knowledge, we can foresee that changing the interaction mode between nanoparticles and proteins (that involved in cell signaling pathways) by physical or chemical ways will provide a way to tune the cell functions.The understanding of the biological properties of CNTs is needed for fully developing their application potentials in biomedicine and for avoiding their nanotoxicity. In the eukaryotic cell, BMP signaling pathway is involved in the regulations of plenty of critical cell functions. In the following parts of this dissertation, we investigated a serial of biological events that are under the controls of this signaling pathway, including stem cell differentiation, cell apoptosis and cell proliferation (cell cycle).The study in the effects of CNTs on stem cell differentiation is important both in clinical practise and in theory. On the one hand, the unique mechanical and electronic properties of CNTs make them a welcome matrix biomaterial in stem cell tissue engineering. Studies showed that their interactions with stem cell fates will determine their performance. On the other hand, the cell signaling pathways that work in stem cells also play a role in some tumor cells. For example, BMP signaling is activated in breast tumor cells although it is originally found to control the differentation of mesenchymal stem cells. So it is safe to say the understanding of CNTs'effects on stem cell differentiation will be illuminating for other application potentials in biomedicine. In the third part of this dissertation (Chapter4and5), we studied the effects of MWCNTs on the myogenic differentiaiton of mesenchymal stem cell C2C12. We found the suppression of BMP signaling pathway by MWCNTs enhanced the myogenic differentiation of C2C12and this effect was subject to fine tuning by the variations in the chemistry on MWCNTs surface.By analyzing the molecular events involved in myogenic differentiation process step by step, we found the inhibition on BMP signaling activity was the reason for enhanced myogenic differentiation of C2C12. The suppression of Id proteins, which were one family of targeted proteins under the regulations of BMP signaling pathway, relieved their inhibiting effects on myogenic regulatory factors, which were members of a protein family with basic helix-loop-helix (HLH) structures. Because we have clarified that MWCNTs bind to type2BMP receptor, and their interaction with proteins are under the controls of their surface characteristics, we hypothesized that changing surface chemistry of MWCNTs will vary this interactions, leading to variable biological readouts including different cell differentiation levels. The screening a combinatory library with different surface chemistry on MWCNTs against myogenic differentiation readout verified this hypothesis, demonstrating human control on nanomaterials'biological properties.We found that the induced overexpression of p21protein by MWCNTs suppressed cell apoptosis and mediated cell cycle arrest in the fourth part of this dissertation (Chapter6and7). p21protein is an inhibitor of both cyclin dependent kinases and cell apoptosis. Our research indicated that the inhibition of MWCNTs on BMP signaling pathway led to the p53-independent overexpression of p21protein. On one hand, p21translocated from nuclei to cytoplasm and bound to Procaspase-3on the mitochondrial membrane, inhibiting the activation of Procaspase-3to active form of caspase-3, thus suppressing cell apoptosis. On the other hand, p21protein deteriorated the functions of CyclinD/CDK4,6complex and inhibited the phosphorylation of Rb protein by this complex, leading to cell cycle arrest.Cell uptake is largely determined by some physical properties of nanoparticles, for example, the dimension and shape. In the last part of the dissertation (Chapter9), we investigated the cell uptake and cytotoxicity of two-dimensional, polymeric nanodisks. The results indicated that changing nanoparticles (NPs) from three-dimensional spherical shape to two-dimensional disk shape greatly reduces their cell uptake without decreasing cell surface binding. They bound on cell membrane and decreased the orders of phospholipid bilayers of cell membrane. As a result of lower cell uptake, nanodisks show very little perturbations on cell functions like cellular ROS generation, apoptosis and cell cycle progression while nanospheres exhibit certain cytotoxicity. Therefore, nanodisks can be a promising template for developing cell membrane-specific imaging nanoagents for a range of biomedicinal applications such as molecular imaging, tissue engineering, in vivo cell tracking, and stem cell separation.
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