| Nanomedicine and nanotoxicity are two important cross-disciplinary fields that were derived from the interdisciplinary and integrated from nanotechnology, life science, medicine and environmental science. In this contribution, we focus on the application of nanomaterials to intracellular protein labeling and tracking as well as its biological toxicity assessment. The mainly points are as follows:1. Aptamer-conjugated gold nanoparticles were developed as optical probes for imaging the internalization pathways of prion protein. By conjugating thiol-modified prion aptamer to the surface of gold nanoparticles, we prepared an aptamer-conjugated nanoparticles probe (Apt-AuNPs) specific to prion protein (PrPC) and then applied the probe for the light scattering imaging and electron transmission microscopic analysis of PrPC in cells. Further investigation showed that caveolin-related endocytosis was likely a necessary pathway for the internalization of PrPC labeled with Apt-AgNPs in human bone marrow neuroblastoma cells (SK-N-SH cells). Owing to its simple preparation and low productive costs, Apt-AuNPs probe offers high promising for further biomedical imaging applications.2. Aptamer adapted silver nanoparticles (Apt-AgNPs) were developed as a novel optical probe for simultaneous intracellular protein imaging and single nanoparticle spectral analysis, wherein AgNPs act as an illuminophore and aptamer as a biomolecule specific recognition unit, respectively. With protein conjugation and aptamer functionalization, AgNPs show satisfactory biocompatibility and stability in cell culture medium. It was found that streptavidin conjugated and aptamer-functionalized AgNPs show satisfactory biocompatibility and stability in cell culture medium, and thus not only can act as a high contrast imaging agent for both dark-field light scattering microscope and TEM imaging but also can inspire supersensitive single nanoparticle spectra for potential intercellular microenvironment analysis. Further investigations showed that caveolae-related endocytosis is likely a necessary pathway for Apt-AgNPs labeled PrPC internalization in human bone marrow neuroblastoma cells (SK-N-SH cells). The integrated capability of Apt-AgNPs to be used as light scattering and TEM imaging agents, along with their potential use for single nanoparticle spectral analysis, makes them a great promise for future biomedical imaging and disease diagnosis.3. A simple and general strategy for specifically protein labeling with nanoparticles is proposed by employing aptamer not only as the identifier for specifically protein recognizing, but also as a linker for targeting streptavidin conjugate nanoparticles (SA-NPs). In our approach, a cell membrane protein is pre-labeled by biotin modified aptamer (Bio-Apt) added to the medium, and then the biotin group serves as a handle for targeting SA-NPs. To demonstrate the feasibility of our strategy, nucleolin, a biomarker of cancer cells, was first labeling and imaging with three kinds of nanoprobes including gold nanoparticles, silver nanoparticles and quantum dots, respectively. Subsequently, single color and double color labeling with both QDs and fluophores to prion protein (PrPC) in neuroblastoma cells were achieved, and then the intercellular distribution of the PrPC as well as their colocalization with transferrin (Tf) were investigated. We further performed time-lapse imaging of QDs bound to PrPC to addressing the dynamic characteristics analysis of different cellular transportation. This new labeling method is simple in procedure, avoiding any complicated probe modification in comparison to traditional approaches. Furthermore, this strategy is generalizable since not only the sequences of aptamers can be changeable for various proteins recognition but also different types of reporters can in principle use for individual or double signal imaging.4. In vivo biodistribution and toxic effects of functionalized QDs in developing zebrafish was systemic investigated through the aquatic exposure and microinjection. Quantum dots are widely explored for biomedical applications, but there is very limited information regarding their in vivo biodistribution and biocompatibility, especially in aquatic animals. Our results demonstrate that multi-PEGylated QDs show weak adsorption capability both on the surface of egg membrane and larval body, and the surface charge of QDs have no effect on such adsorption. However, carboxylated quantum dots which exposure in aquatic environment, can not only adsorbed on the embryonic chorion in large numbers, but also effectively accumulated in the tissues of larvae, distributing in oropharyngeal cavity, gills, fins and anus. Furthermore, QDs can move easily into the digestive canal through the role of larval swallowing. High concentrations of carboxylated quantum dots exposure can induce abnormal embryonic development that may be caused by the release of metal cadmium. When introduced into the circulation system by heart microinjection, carboxylated quantum dots will be transported into the venous system through blood circulation of zebrafish, mainly distributing in the intravenous after brain, nasal orbital vein, heart, abdominal wall, spinal vein, as well as tail artery and vein. What is more important, a large number of carboxylated quantum dots still residue within the vein and were not cleaned out by the body at 6 days after the injection, showed a high adsorption capacity and in vivo biological residue, suggesting the high potential in biological toxicity.5. A comparative study of nano-toxicological effect to graphene and mutli-walled carbon nanotubes (MCNTs) was achieved through model organisms exposed methods. Graphene is a new type of carbon nanomaterials that has stimulated great interest due to their superior mechanical, electrical, and thermal properties. However, no special toxic assessment of graphene, so far, has been reported as far as we know. Our results demonstrate that graphene would cause obvious cell growth inhibition and slight hatching delay of zebrafish embryo at a concentration of 50 mg/L, but can not lead to increased apoptosis in embryonic and serious fetal malformations, showing a moderate toxicity. In comparison to mild toxicological effect of graphene, MCNTs exhibits acute toxicity to organisms. MCNTs can obviously inhibit cell growth at the concentration of 7.6 mg/L, and heavy accumulate in embryonic chorion at 50 mg/L, resulting in significantly increasing of apoptosis in embryo that eventually lead to fetal malformation. These results demonstrate that both graphene and MCNTs have shown toxic effect under certain condition. It is worth noting that graphene and MCNTs, while having a similar nano-morphology, exhibit quite different toxic effects, this may be derived from their distinctive interaction mode to the organisms.
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