奈米科技快速的發展使得各種奈米材料商品已經充斥在生活中，而這些奈米級材料對人類及環境可能造成的危害也越來越受重視。
本研究首先利用低壓式電子衝擊器與雷射奈米粒徑分析儀探討奈米氧化鋅粒子在大氣與水體環境中的分布及凝聚特性，並比較不同方式之超音波處理對於水體中奈米粒子分散性的影響。其後也針對不同pH值、離子強度、界面活性劑之存在等條件測試奈米氧化鋅的聚集特性。進而探討奈米氧化鋅於更複雜的生物培養液中的懸浮與沉澱特性，將結果利用於找出適合的暴露環境以評估奈米氧化鋅之細胞毒性。另一方面，本研究也藉由自製奈米微粒探討奈米氧化鋅的物化特性 (大小、表面積、形狀、表面結構) 對於細胞毒性的影響，最後則藉由在奈米氧化鋅上被覆二氧化鈦來探討其被覆厚度與被覆物晶型對於細胞毒性的影響。
結果發現當奈米粒子自空氣進入水體中後會形成嚴重的凝聚現象，而經由不同方式的超音波處理後，發現以探針式的處理分散效果最好，經處理過的粒徑分布與由電子式低壓衝擊氣所偵測之氣動粒徑範圍非常相近。而水體的離子強度、酸鹼度和界面活性劑的添加對於奈米氧化鋅的凝聚特性產生相當大的影響，其原因則歸咎於粒子表面電雙層厚度之變化。然而當奈米氧化鋅進入生物培養液(DMEM)後，即使經過超音波處理其水動粒徑仍高達900 nm，且高濃度的奈米氧化鋅懸浮液由於粒子間接觸頻率高，導致奈米粒子的凝聚與沉澱速率皆增加。如在生物培養液中加入不同濃度(0.5-10%) 的胎牛血清，則能有效改善其分散性；且在實際的MTT及IL-8細胞毒性測試中也發現以不含胎牛血清的培養液暴露奈米氧化鋅粒子最能在較短時間呈現其毒理效應。本研究使用沉澱法及醇熱法製備不同大小的桿狀與球形奈米氧化鋅，也藉由添加油酸使微粒表面呈現疏水性。進行毒性試驗後發現粒徑較小的奈米氧化鋅無論在桿狀或球形的毒性都較高，而桿狀奈米氧化鋅的毒性在相似的粒徑下又比球形奈米氧化鋅之毒性強。比較經油酸處理前後之奈米氧化鋅毒性，由於處理後奈米氧化鋅之疏水特性導致其毒性減弱，但由IL-8的測定顯示此材料本身可誘發出比未經油酸處理之奈米氧化鋅更多的趨炎物質。研究也發現，雖然奈米微粒的表面積大小影響其毒性，但並非最主要因素。
在奈米殼核體的研究結果部分，經由分析鑑定後，證實製備時間與二氧化鈦的被覆厚度有關，且發現被覆之二氧化鈦之形態為非晶形。將此非晶形純二氧化鈦與商業化二氧化鈦( Degussa P25與ST-21) 之毒性比較後發現非晶形二氧化鈦具有較顯著之細胞毒性。由此可知奈米粒子之晶型和其細胞毒性也有相當程度的關係。雖然二氧化鈦仍然具有細胞毒性，其殼層厚度對於奈米氧化鋅毒性的減弱具有一定的貢獻，其主因則推測是二氧化鈦殼層減緩鋅離子釋放的速率所造成。
綜合上述，本研究可觀察到在相同濃度的條件下，奈米微粒物化特性對細胞毒性影響效應大小如下:
物種 > 晶相 > 形狀 > 大小 > 表面積

The rapid development of nanotechnology makes various kinds of nano-commodities have been packed in our life, but these nano-materials which may cause hazards to human and environment also lead much attention.
In this study, the electrical low pressure impactor (ELPI) and Zetasizer were first used to discuss the different size distribution and agglomerate state of nano-ZnO in various sizes. Different sonication methods were compared for improving the dispersion of nanoparticles in water. Then, the agglomeration state of nanoparticles in various conditions water sysem such as pH value, ionic strength and existence of surfactant were discussed. Another study focused on the agglomeration and sedimentation of nanoparticles in a complicated DMEM cell culture medium. The results would be estimated suitable conditions for assessing the nano-ZnO cytotoxicity. In addition, self-prepared nano-ZnO which had different physicochemical properties (sizes, shapes and surface properties) were utilized to assess their correlation with toxicological responses. Finally, TiO2-coated ZnO was prepared to investigate the shell thickness effect and phase effect on the cytotoxicity.
The results showed that when nano-ZnO transported from air to the water, the particles aggregated seriously. After comparing different sonication methods, the probe sonication got excellent dispersion result. The pH value, ionic strength and the surfactant existence of water contributed significant effect on the hydrodynamic size of nanoparticles, which was attributed to the variety of double electric layer on the particles surface. As particles dispersed to DMEM medium, the hydrodynamic size of particles increased to 900 nm even the suspension was sonicated. If serum was added into the medium, the suspension became stable. In MTT and IL-8 experiments, it could find that particles appeared more toxicity in serum-free medium.
Nanorod and nanosphere ZnO were successfully produced by precipitation and solvothermal methods, the hydrophobic surface property of nano-ZnO was also prepared by adding oleic acid. Smaller particles had more toxicity than the large one could be found in either rod and sphere particles. Furthermore, under similar particle size the rod shapes ZnO were more toxic than sphere shapes. Hydrophobic ZnO particles in water system caused serious agglomeration and lower toxicity, while the more amount of potential IL-8 release than the non-modified nanoparticles. Interestingly, the particle surface area didn’t show significant relations on its toxicity.
The ZnO-TiO2 core-shell structure was prepared and confirmed that increasing coating time increased shell thickness. The amorphous TiO2 shell was demonstrated by XRD, and this material exhibited more potential toxicity than other phases TiO2 nanoparticles (P25 and ST-21), which represented the phases of nanoparticles played more important role than their size effect. Though amorphous TiO2 still had toxicity, the thickness shell slightly decreased the toxicity of nano-ZnO. It might be attributed to the lower rate of releasing zinc ion or the partial uncovered ZnO.
In this study, under the same mass concentration dosage, the effects of different physicochemical properties of nanoparticles inducing the toxicological responses are prioritized as follows: species > phase > shape > size > surface area.

Abstract I
摘要 II
Contents III
List of Tables VII
List of Figures VIII
Chapter 1 Background and Introduction 1
1.1 Nanotoxicology-smaller is not always better 1
1.2 In vitro cytotoxicology 2
1.3 Objects of this study 2
1.4 References: 4
Chapter 2 Literature review 6
2.1 Nanotechnology 6
2.2 Nanomaterials 7
2.3 Nano-ZnO 8
2.3.1 Physico-chemical properties and its applications 8
2.3.1.1 Chemical properties 8
2.3.1.2 Crystal structure 9
2.3.1.3 Electronic properties 10
2.3.1.4 Applications 10
2.3.2 Exposure routes 12
2.3.3 Methods to synthesize nano-ZnO 13
2.4 Concept of nanoparticles cytotoxicity 14
2.4.1 Cytotoxicity assays 14
2.4.2Human lung carcinoma epithelial cell line 16
2.4.3 The role of physicochemical characteristic on biological response 18
2.4.3.1 Particle size 19
2.4.3.2 Dispersion factors 20
2.4.3.3 Surface area 22
2.4.3.4 Surface charge 23
2.4.3.5 Chemical composition 24
2.4.3.6 Shapes 25
2.4.3.7 Solubility 25
2.4.4 Possible mechanism by which nanomaterials interact with biological tissue 26
2.4.5 In vitro toxicity studies of Nano-ZnO 29
2.5 Concept of core-shell colloidal nanoparticles 30
2.5.1 Inorganic and composite coatings 31
2.5.2 In vitro toxicity of core-shell nanoparticles 32
2.6 References 33
Chapter 3 Experimental procedures 42
3.1 Chemicals and solutions 42
3.2 Nano-ZnO commercial nanopowder collection 44
3.2.1 ELPI 44
3.2.2 Collection system 45
3.2.3 Collected powder disperse from filter to water 46
3.3 Nano-ZnO nano powder disperse in water 46
3.3.1 Sonication methods study 47
3.3.2 Concentration study 47
3.3.3 Temperature study 47
3.3.4 pH and ionic strength study 47
3.3.5 Surfactant study 48
3.3.6 Relations between particles size and zeta potential 48
3.4 Nano-ZnO nano powder disperse in DMEM cell culture medium 48
3.4.1 Concentration study 48
3.4.2 Protein study (serum) 49
3.4.3 Various primary size of as-prepared nanorods ZnO study 49
3.5 Interference of nano-ZnO on cytotoxicity assay 50
3.5.1 Absorption effect 50
3.5.2 Adsorption effect (IL-8) 51
3.6 Preparation of different size, shape and surface structure nano-ZnO 51
3.6.1 Prepare various sizes ZnO nano-rod 51
3.6.2 Prepare various sizes ZnO nano-sphere 51
3.6.3 Prepare monodispersed sphere nano-ZnO 52
3.6.4 Preparation of TiO2-ZnO core-shell nanoparticles 52
3.6.5 Nomenclature and preparation conditions of self-prepared nano-ZnO 53
3.7 Nanoparticles characterization 54
3.7.1 TEM 54
3.7.2 XRD 55
3.7.3 Zetasizer 56
3.7.4 BET 57
3.7.5 UV-vis spectrophotometer 58
3.7.6 ICP-AES 59
3.7.7 Raman spectroscopy 59
3.7.8 XAS (XANES, EXAFS) (data see appendix II) 60
3.8 Cytotoxicity and inflammation studies of nano-ZnO particles on A549 cell lines 61
3.8.1 Cell culture 61
3.8.2 Preparation of stock solution 61
3.8.3 Sample treatment 62
3.8.4 Cell morphology 62
3.8.5 MTT assay 62
3.8.6 Trypan blue exclusion assay 63
3.8.7 Production of IL-8 63
3.8.8 Statistics 64
3.9 References 64
Chapter 4 Size distribution and agglomeration effect on nano-ZnO dispersion in air and aqueous solution 65
4.1 Size distribution difference between air and aqueous surroundings 66
4.2 The effect of different sonication methodologies in preparing dispersions 69
4.3 Dispersion state of nano-ZnO in different conditions of solutions 70
4.3.1 Concentration study 70
4.3.2 Temperature effect 72
4.3.3 Ionic strength and pH effect 73
4.3.4 Surfactant effect 79
4.3.5 Particle size effect on zeta potential 80
4.4 Summary 82
4.5 References 83
Chapter 5 Behaviors of size, agglomeration and sedimentation of nano-ZnO in cell culture medium and its toxicological studies 85
5.1 Agglomeration and sedimentation studies for ZnO nanoparticles in DMEM 85
5.1.1 Size distribution difference of nano-ZnO between water and DMEM medium 85
5.1.2 Concentration study 87
5.1.3 Protein study 89
5.1.4 Size study 92
5.2 Nano-ZnO toxicity studies under serum or serum-free medium 93
5.2.1Nano-ZnO particles properties 93
5.2.2 Cell morphology 94
5.2.3 MTT and IL-8 assay with or without serum exposure 96
5.3 Particles interference in toxicological studies 99
5.3.1 Particles absorption effect for MTT assay 99
5.3.2 Protein adsorption effect on IL-8 assay 100
5.4 Summary 102
5.5 References 103
Chapter 6 Physico-chemical characters of prepared nano-ZnO play an important role on the toxicity of human lung epithelial cells 105
6.1 Characterization of Nano-ZnO Samples 105
6.1.1 Nanorod ZnO particles 105
6.1.2 Nanosphere ZnO particles 110
6.1.3 Modified sphere ZnO nanoparticles 113
6.2 Cytotoxicity and inflammatory mediator results with A549 cell 115
6.2.1 Size factor 115
6.2.2 Shape factor 120
6.2.3 Surface area factor 122
6.2.4 Surface modification factor 123
6.3 Discuss the possible mechanism for ZnO toxicity in A549 cells 126
6.4 Summary 126
6.5 References 127
Chapter 7 In vitro toxicity of ZnO-TiO2 core-shell nanoparticles 129
7.1 Characterization of ZnO-TiO2 core-shell nanoparticles 129
7.1.1 XRD and TEM 129
7.1.2 IR and Raman 133
7.1.3 UV-vis 135
7.1.4 DLS (hydrodynamic size and zeta potential) and ICP-AES 137
7.2 Cytotoxicity of TiO2 shell: phase effect 138
7.2.1 Characterization of Nano-TiO2 Samples 138
7.2.2 Cytotoxicity and inflammatory mediator results with A549 139
7.3 Cytotoxicity of ZnO-TiO2 core-shell nanoparticles 141
7.3.1 Cytotoxicity of ZnO-TiO2 and comparative physical mixture particles 141
7.3.2 Shell effect 143
7.4 Summary 146
7.5 References 147
Chapter 8 Conclusions 149
Appendix 151

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