Study Of Size-dependent Biological Effects And Applications Of Nanomaterials

The word of nanotechnology has drown our great attention since the concept of“nano” was brought up in the middle of last century. Nanometer is actually a unit oflength, one nanometer is equal to one billion of one meter. Nanotechnology refers toinvestigating and exploring the property and applications of materials, whose size wasin the range from0.1nm to100nm. It is the unique size of the nanomaterials that givesthem the special effects (such as small size effect, large surface effect, quantumtunneling effect, etc.) and the broad application prospect. In recent years, more andmore attentions were paid to the biomedical applications of nanomaterials, and thisfield is growing rapidly. Researchers kept developing new, special, andmultifunctional materials for target delivery, biosensing, bioimaging and biocodingetc. As the study goes deeper, people started to realize the importance of the size, eventhe same material in the same nanoscale, the size will show great impacts on itsfollowing bioapplications.This thesis selected two typical systems as representatives, one is the well-knowntwo dimensional graphene oxide nanosheets, and the other is DNA nanostructures, hotstudied in recent years. We first designed and prepared them with controllable sizes,then we did a serials of characterization on them to ensure our following studies. Next,we made our great efforts to explore their size-dependent biological effects andapplications, mainly including cytotoxicity, cellular uptake behaviors andinternalization mechanisms, biosensing and antibacterial applications. The mainresearch content and results are summarized as follows:(1) Preparation and characterization of different sized graphene oxide nanosheets.In this work, we developed a high yield, low-cost, convenient method via severalrounds of oxidation for preparing three different sized GO nanosheets (GO-1, GO-2, GO-3) based on modified Hummers’ method. AFM (atom force microscopy)images showed that the height of three prepared GO nanosheets (GO-1, GO-2, GO- 3) was about1.1nm, indicating that they are all one atomic thick. TEM(transmission electron microscopy) and DLS (Dynamic light scattering) resultsshowed that the average lateral size of GO-1, GO-2, and GO-3was500nm,200~300nm, and30~50nm separately. XPS (X-ray Photoelectron Spectroscopy)showed that the smaller the GO nanosheets was, the higher degree of oxidizationit owned.(2) Study of size-dependent biological effects and applications of graphene oxidenanosheets.The results showed that the cytotoxicity of graphene oxide was size-dependent,smallest sized nanosheets exhibited lowest cellular cytotoxicity. Besides, threesized graphene oxide nanosheets (GO-1, GO-2, GO-3) were labelled byradioactive125I to determine the amount of intracellular uptake by isotopelabelling method. Our results showed that the cellular uptake of graphene oxidenanosheets were obviously size-dependent, and the ultra-small sized GO-3nanosheets had the highest uptake amount. Based on the two results, we believethat the GO-3has the best prospects for cell biology applications. Moreover,according to our previous report of excellent antibacterial property of grapheneoxide, we compared the antibacterial activities of the as mentioned three GOnanosheets. Results showed that the antibacterial activity of graphene oxidenanosheets was size-dependent too, and the larger the size of GO, the betterthe antibacterial property.(3) Size-Dependent Programming of the Dynamic Range of Graphene Oxide–DNAInteraction-Based Ion SensorsWe found that the the fluorescence quenching ability of GO nanosheets towardsfluorophore labelled single strand DNA was size-dependent, and the quenchingefficiency was inversely proportional to the length of single-stranded DNA. Thequenching effect of GO3(~30nm) was worst, comparing with the other two GOnansheets. We mainly contributed this to the differnent interactions between GOnanosheets and single strand DNA. Based on the previous results, we designed aseries of programmable dynamic range of graphene oxide–DNA interaction-based ion sensors for Hg2+. Sensor based on GO1demonstrated broad dynamic rangefrom1nM to40nM. Sensor based on GO2demonstrated dynamic range from1nM to15nM. Sensor based on GO3demonstrated narrow dynamic range from0.1nM to5nM.(4) Design, synsthesis and characterization of one dimensional DNA nanostrings withcontrollable length.With the rapid development of DNA nanotechnology, DNA nanostructures withunique advantages have gained much attention in the biomedical area. However,the interactions between DNA nanostructures and cell were not clear, and study ofcellular uptake behaviors of DNA nanostructures was quite essential for the furtherbiological applications of DNA nanostructures. Hence, we firstly desingned twobuilding blocks (TH1, TH2), and prepared a serials of DNA nanistrings withdifferent length based on hybridization chain reaction. Besides, the length wastuned by controlling the relative ratio of THs and the initial strand (Hi). The AFMiamges showed that the average length of TH1,1-pair,5-pair, and10-pair DNAnanostring was about15nm,32nm,160nm, and330nm respectively. Next, westudied the extracellular stabilities of these DNA nanostructures by Nativepolyacrylamide gel electrophoresis and fluorescence resonance energy transfer(FRET). The results showed that our DNA nanostructures can keep intact for ateast12h under our experimental condition, which was prerequisite for ourfollowing studies of their cellular uptake behaviors.(5) Study of size and environmental-dependent cellular uptake behaviors andinternalization mechanisms.The results showed that the cellular internalization of the as-mentioned DNAnanostructures was related to their size and their cell incubation conditions. Wefound that the internalized quantity of DNA exhibited a positive relation with thesize of DNA nanostructures in FBS free medium, and Nanostructures withrelatively larger sizes (5-pair and10-pair DNA nanostring) showed much highercellular internalization. However, in the case of10%FBS-containing mediumincubation condition, things were quite different. The internalization of DNA nanostructures exhibited a wave-like alteration, and the uptake of TH1DNAnanostructure was significantly higher than other DNA nanostructures. We thenproceeded to further investigate the exact internalization patheways DNAnanostructures used by using a series of known biochemical inhibitors. As theresults showed, we believe that DNA nanostructures in our studies mainly entercells through phagocytosis or macropinocytosis pathway under FBS-freecondition. When it comes to10%FBS-containing condition, pathways showedmuch diversity, mainly related to their size, but all were through receptor-mediatedendocytosis internalization pathway. Then, we exploring the reason why theinternalization pathways of DNA nanostructures were so different under differentincubation condition using microscale thermophoresis. Our results showed thatthere are weak interactions between FBS and DNA nanostructures and the TH1had the strongest interaction, which could well explain our previous results ofdifferent cellular uptake behaviors.Though systemastically investigating one-demisonal and two-demisionalnanomaterials, we found that the biological effects and applications ofnamomaterials were size-dependent. These findings were quite helpful in designingspecific nanocarriers, nanoprobes and biosensors by taking full advatages ofdifferent sized nanomaterials and benifited the biolomedical applications ofnanomaterials.