臺灣博碩士論文加值系統

近年來，奈米材料應用在生物醫學中的影像分析、藥物傳送和治療是持續被發展的課題。在本研究中，我們結合了二氧化鋅奈米粒子的二倍頻訊號和皮膚角質細胞的螢光訊號來觀察二氧化鋅奈米粒子在化學促進劑如:油酸、乙醇和油酸-乙醇的影響下，在皮膚的穿透行為。除了分析本質上的影像結構，二氧化鋅穿透的特性也同樣的被定量分析，而得到載體對皮膚的分佈係數、二倍頻訊號的強度梯度和有效碰撞路徑長度。這些結果顯示油酸、乙醇和油酸-乙醇能夠有效的增加二氧化鋅奈米粒子在皮膚的穿透深度，是因為增加了皮膚皮酯的流動性或是改變了皮膚角質層的皮酯排列緊密性。
更進一步的，不需要額外的染色步驟，當眼角膜上表皮保護層受到損害後，利用雙光子顯微術也可以觀察到螢光奈米粒子穿透眼角膜和滯留在眼角膜細胞間。在細胞毒性實驗中，我們使用牛眼角膜基質細胞和奈米粒子做細胞培養，可以發現細胞的存活率會隨著奈米粒子的濃度增加和培養時間的增長而有明顯的減少。並且，在老鼠動物實驗中，雙光子顯微術影像顯示出奈米粒子可以滯留在眼角膜中達到26天以上。根據在細胞跟動物實驗所得到的實驗結果，我們推測，當眼角膜的上表皮保護層受到損壞後，奈米粒子可以穿透並長時間滯留在眼角膜中，而對細胞造成毒性。
奈米材料應用在藥物傳遞方面，我們也合成金奈米棒的藥物複合體。金奈棒藥物複合體是將金奈米棒、目標藥物和螢光分子，用電解質聚合物包覆起來。合成好的金奈米棒藥物複合體，在飛秒紅外光雷射照射下，我們也詳細的研究了被釋放螢光分子的藥物動力學。螢光分子會因為吸收了由金奈米棒將紅外光雷射轉換而來的熱，而從金奈米棒的藥物複合體中釋放出去。釋放出去的螢光分子則在紅外光雷射連續性照射和周期性照射兩種不同模式下測量。在照射紅外光雷射時間為五分鐘時，螢光分子的釋放速率在雷射連續性照射和周期性照射下，分別呈現零級和一級的動力學機制。更進一步，我們也設計了金奈米棒藥物複合載體，用電解質聚合物包埋了金奈米棒和抗癌藥物太平洋紫杉醇而形成藥物載體。抗癌藥物太平洋紫杉醇可以用雷射誘導而從金奈米棒複合體中釋放出去。而釋出的抗癌藥物太平洋紫杉醇對乳癌細胞的細胞抑制率則和紅外光雷射的照射方式及照射時間有關。

The biomedical applications of nanomaterials in imaging, drug delivery, and therapy have led to ever-growing developments in the past decades. In this work, we combined the second harmonic generation of ZnO nanoparticles and the autofluorescence of the stratum corneum to image the penetration of ZnO nanoparticles under the chemical enhancer conditions of oleic acid, ethanol and oleic acid-ethanol. In addition to qualitative imaging, the microtransport properties of ZnO nanoparticles were quantified to give the enhancements of the vehicle-to-skin partition coefficient, the second harmonic generation intensity gradient and the effective diffusion path length. The results showed that oleic acid, ethanol and oleic acid-ethanol were all capable of enhancing the transdermal delivery of ZnO nanoparticles by increasing the intercellular lipid fluidity or extracting lipids from the stratum corneum.
Furthermore, with no additional staining, the two-photon image showed that fluorescent nanoparticles penetrated and resided within interlamellar space of cornea stroma when corneal epithelium barrier was injured. In vitro cytotoxicity test using bovine corneal stromal cells incubated with nanoparticles indicated that the cell viability decreased significantly as the nanoparticles concentration and incubation period increased. Moreover, two-photon imaging showed that nanoparticles can retain within cornea up to 26 days in an in vivo mouse model. On the basis of our in vivo and in vitro data, we conclude that nanoparticles can penetrate and retain within cornea long enough to cause consequential cytotoxicity, under the circumstance that corneal epithelium barrier is injured.
In drug delivery applications of nanomaterials, the conjugates of gold nanorods and the model drug, fluorescein isothiocyanate (FITC), embedded inside polyelectrolytes (GNRs/FITC@PLE) were synthesized to study the release kinetics of FITC under femtosecond near-infrared (NIR) laser irradiation. The release of FITC from the conjugates was induced by the heat generated from gold nanorods under laser irradiation. The concentration of released FITC was measured as the time of continuous and periodic laser irradiation was varied. Within 5 min of the laser exposure, the release rates of FITC exhibited zero-order and first-order kinetics under continuous and periodic irradiation, respectively. Furthermore, a drug release system was designed based on the conjugates of gold nanorods and the anticancer drug, paclitaxel (PTX), embedded inside polyelectrolytes (GNRs/PTX@PLE). The release of PTX from the conjugates was triggered by NIR laser irradiation, and the inhibition rates of breast cancer cells showed strong dependencies on the irradiation modes and time.

總目錄
摘要 I
Abstract III
總目錄 V
Part I
Chemical Enhancer Induced Changes in the Mechanisms of Transdermal Delivery of Zinc Oxide Nanoparticles
1.1 Abstract 1
1.2 Introduction 2
1.3 Experimental Section 4
1.3.1 Preparation of ZnO Nanoparticles 4
1.3.2 Donor Solution Preparation 4
1.3.3 Preparation of the Skin Samples of Nude Mice for Multi-photon Microscopy Imaging 5
1.3.4 Dual-channel Multi-photon Microscopy 6
1.3.5 Data Collection and Analysis 6
1.4 Results and Discussion 8
1.4.1 Optical Properties, Dual-channel Two-photon and Electron Microscopic Images of ZnO Nanoparticles 8
1.4.2 Imaging the Chemical Enhanced Transdermal Delivery of ZnO Nanoparticles 10
1.4.3 3-D SHG Images of ZnO NPs Distribution in the SC under Different Chemical Enhancer Conditions 13
1.4.4 Axial SHG Intensity Profiles of ZnO Nanoparticles 15
1.4.5 Chemical Enhancements in the Vehicle-to-skin Partition Coefficient, the Intensity Gradient and the Effective Diffusion Path Length for ZnO Nanoparticles 16
1.5 Conclusion 18
1.6 References 19

Part II
Studies of Intracorneal Distribution and Cytotoxicity of Quantum Dots: Risk Assessment of Eye Exposure
2.1 Abstract 22
2.2 Introduction 22
2.3 Experimental Section 26
2.3.1 QDs Capped with Different Functional Groups 26
2.3.2 Bovine Cornea Incubated with QDs for Two-photon Microscopy Imaging 26
2.3.3 In Vivo Intrastromal QD Injection in the Mice Model 27
2.3.4 Cell Culture and QDs Treatment for BCF and BSF 27
2.3.5 MTT Cell Viability Assay 28
2.3.6 Measurement of QD Uptake Using Flow Cytometry 28
2.3.7 Statistical Analysis of Data 29
2.4 Results and Discussion
2.4.1 Permeability and Distribution of QDs through the Cornea 29
2.4.2 In Vivo Corneal Bioaccumulation of QDs 34
2.4.2 Cytotoxicity of QDs in Vitro 35
2.4.3 QD Uptake in the BCF 38
2.5 Conclusions 39
2.6 References 40

Part III
Multiple Release Kinetics of Targeted Drug from Gold Nanorod Embedded Polyelectrolyte Conjugates Induced by Near-Infrared Laser Irradiation
3.1 Abstract 46
3.2 Introduction 47
3.3 Experimental Section 50
3.3.1 Materials 50
3.3.2 Synthesis of Gold Nanorods 50
3.3.3 Preparation of GNRs/FITC@PAA Conjugates 51
3.3.4 Preparation of GNRs/PTX@PDC 52
3.3.5 Preparation of GNRs/PAA@PDC 53
3.3.6 FITC Released from GNRs/FITC@PAA Conjugates Using Femtosecond NIR Laser Irradiation 53
3.3.7 Cell Viability Assays of MCF-7 Cells after Incubation with Gold Nanorods or GNRs/PTX@PDC or GNRs/PAA@PDC Conjugates 54
3.3.8 Cell Viability Assays of MCF-7 Cells Incubated with GNRs/ PTX@PDC or GNRs/PAA@PDC Conjugates after NIR Irradiation 55
3.4 Results and Discussion 56
3.4.1 Structural Properties of Gold Nanorod Conjugates before and after Laser Irradiation 56
3.4.2 Adjustments of the Power and Time of Laser Irradiation on Gold Nanorod Conjugates Based on UV-vis Spectroscopy and TEM Measurements 57
3.4.3 Release Kinetics of FITC from GNRs/FITC@PAA Conjugates by Continuous NIR Laser Irradiation 60
3.4.4 Release Kinetics of FITC from GNRs/FITC@PAA Conjugates by Periodic NIR Laser Irradiation 62
3.4.5 Mechanism of FITC Released from GNRs/FITC@PAA under Laser Irradiation 65
3.4.6 Fluorescence Image of GNRs/FITC@PAA Conjugates in Cells Using Confocal Microscopy 67
3.4.7 Cell Viability Assay of GNRs/PTX@PDC Conjugates Incubated with MCF-7 Breast Cancer 68
3.4.8 Inhibition Rates of GNRs/PTX@PDC Conjugates for MCF-7 Breast Cancer Cells after NIR Laser Irradiation 69
3.5 Conclusions 75
3.6 References 75

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