伴隨著納米科技的發展，越來越多的納米顆粒系統已經應用於生物醫學领域。其中，二氧化硅納米顆粒因其簡單易操作的表面化學性質和在生理環境中的良好穩定性，已被廣泛認為是最有前途的治療和診斷載體之一。 / 在本論文中，我首先對二氧化硅納米顆粒與人類細胞之間的作用進行了系統研究。這些作用包括了以下主要特點: 胞吞和胞吐被分別確定為納米顆粒主要的進入和離開細胞的主要途徑; 大部份的納米顆粒被發現存在於有膜結構的細胞器里，這些細胞器相當穩定（不易破損），只有很少的一部份納米顆粒被釋放到細胞質里; 納米顆粒和細胞之間的作用是動態的，它們進入細胞內的數量由其在細胞培養液中的數量和形態（聚集的程度）所決定。正是這些特點決定了二氧化硅顆粒在低濃度時的低細胞毒性。 / 緊接著我比較了兩種最常見的二氧化硅納米顆粒（晶體態和無定型態）引入細胞后對細胞所帶來的影響。儘管兩種形態的納米顆粒所造成的細胞毒性都比較低，但是更細緻的分析揭示了它們對細胞及其衍化途徑的不同影響。細胞吞入晶體態的二氧化硅納米顆粒后，其內部的活性氧物質含量顯著提高，這種變化會導致細胞線粒體功能受損（表現為線粒體增生）並且最終將細胞導向死亡。不過只有在p53基因缺失的細胞中才有這種由活性氧物質水平升高導致的細胞損傷，p53正常的細胞卻能抵禦這種來自晶體態二氧化硅納米顆粒的刺激。而無定型態二氧化硅納米顆粒對生物系統無損害，因而有發展為藥物載體的巨大潛力。 / 基於對二氧化硅顆粒細胞毒性研究的理解，我們設計了一種新型納米載體--金核/二氧化硅殼層（Au@SiO₂）納米顆粒用於藥物輸運。在這一體系中，無定形態二氧化硅和金納米顆粒的優勢被整合在一起，同時光敏劑（PS）藥物分子被裝載在二氧化硅殼層內。對比於自由形式的PS，裝載在Au@SiO₂納米顆粒中的PS展示出增強的藥效。需要強調的是，用這種納米顆粒處理的細胞以阻梗壞死為主要的死亡途徑，代替了凋亡這種不太有效的方式。在光照下，金的等離子體效應被發現能促進PS的光響應過程，這使得細胞殺死率得到了大幅度增強。這一效應得益于我們把PS束縛在金核的表面，同時保證金表面等離子體振盪能量和PS吸收能量的配對。此外，把PS裝載在二氧化硅中會引起PS有益的光化學改變。這些作用結合在一起導致了藥效的提高。這些機理能被普遍應用於納米顆粒裝載藥物分子的設計中，為最優化設計提供指導。 / With recent development of nanotechnology, various nanoparticulate systems have been proposed to serve as functional units for biomedical applications in many innovative ways. Among various possible choices, silica nanoparticles (NPs) enjoys easily modifiable surface chemical characteristics and excellent stability in physiological environment. Therefore, it is considered as one of the most promising carrier candidate for therapeutic and diagnostic applications. / A systematic study on the interaction between silica nanoparticles and human cells is first carried out in the present thesis work. Endocytosis and exocytosis are identified as major pathways for NPs entering, and exiting the cells, respectively. Most of the NPs are found to be enclosed in membrane bounded organelles, which are fairly stable (against rupture) as very few NPs are released into the cytoplasma. The nanoparticle-cell interaction is a dynamic process, and the amount of NPs inside the cells is affected by both the amount and morphology (degree of aggregation) of NPs in the medium. These interaction characteristics determine the low cytotoxicity of SiO₂ NPs at low feeding concentration. / Experiments were then designed to compare the the biological consequence of two most common form of SiO₂ nanoparticles, i.e., crystalline and amorphous NPs, when they were introduced to human cells. Although the apparent cytotoxicity of both types of NPs seems to be low, more detailed characterizations disclose the profound difference induced by the crystalline and amorphous ones, resulting in significantly different cell evolution pathways. Crystalline NPs but not amorphous ones are found to drastically increase the recative oxygen species (ROS) level in the cells, which can cause mitochondria dysfunction (being expressed as mitochondria proliferation), and eventually direct the cell into apoptosis. Nonetheless, only p53 deficient cells are subjective to such ROS induced cell damage, while p53 proficient cells can accommodate the stimulation from crystalline SiO₂ NPs. The amorphous SiO₂ NPs are found to be benign in the biological systems, and have great potential to be developed as nanomedicine. / Base on the understanding obtained from the toxicology study of the SiO₂ NPs, we have designed a special nanocarrier system for drug delivery. We have combined advantages of both SiO₂ and Au NPs by constructing Au-core/SiO₂-shell (Au@SiO₂) nanocarriers with the photosensitizer (PS) drug embedded in the SiO₂ shell layer. Compared with free PS, PS loading in the Au@SiO₂ NPs showes a enhanced drug efficacy. In particular, the cells treated with the NP drug take necrosis as a major death path instead of apoptosis, which is a much less effective route. The Au plasmonic effect is found to promote the photo-response of the PS drug under light irradiation, contributing to the largely decreased cell viability. Nevertheless, one shall note that spatial confinement of the drug moledules to the close proximity of the Au core and an energy match between the drug absorption and the Au surface plasmon resonance are critical in manifesting the plasmonic effect. At the same time, embedding the drug in the SiO₂ matrix leads to favorable change in the photochemical process. The combined effects brought by the Au@ SiO₂ NP carrier is responsible for the high drug efficacy. These mechanisms can be generally valid in engineering drug molecule incorporation into NP carriers and also give guidance for the optimum design of the NP drug carrier. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chu, Zhiqin = 二氧化硅納米顆粒與人類細胞的作用及其在生物醫學方面的應用 / 褚智勤. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 120-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chu, Zhiqin = Er yang hua gui na mi ke li yu ren lei xi bao de zuo yong ji qi zai sheng wu yi xue fang mian de ying yong / Chu Zhiqin. / Table of contents --- p.VIII / List of figures --- p.XIII / List of tables --- p.XIX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Background --- p.4 / Chapter 2.1 --- Overview of the silica-based nanoparticles for bio-medical applications --- p.4 / Chapter 2.2 --- Health issue on the silica-base nanoparticles --- p.5 / Chapter 2.3 --- Understanding the nano-bio interface --- p.6 / Chapter 2.3.1 --- Nano-bio interface in vitro --- p.7 / Chapter 2.3.2 --- Nano-bio interface in vivo --- p.10 / Chapter 2.4 --- Bio-application of silica-based nanoparticles --- p.11 / Chapter 2.4.1 --- Use of silica nanoparticle as imaging agent --- p.11 / Chapter 2.4.2 --- Use of silica nanoparticle as drug carrier --- p.12 / Chapter 2.4.3 --- Use of silica nanoparticle as coating media --- p.12 / Chapter 2.5 --- Surface plasmon of gold nanostructures and its bio-application --- p.13 / Chapter 2.5.1 --- Introduction to the SPR of gold nanostructures --- p.13 / Chapter 2.5.2 --- Synthesis of gold NRs and their SPR effect --- p.13 / Chapter 2.5.3 --- SPR of gold NRs in bio-application --- p.16 / Chapter Chapter 3 --- Experimental --- p.18 / Chapter 3.1 --- Standard methodologies for nanoparticle preparation and their feeding to the cells --- p.18 / Chapter 3.2 --- Cell sampling for room temperature TEM study --- p.18 / Chapter 3.3 --- Developing methods to distinguish NPs in cell sample under TEM --- p.20 / Chapter 3.4 --- Confocal microscopy study --- p.21 / Chapter 3.4.1 --- Study the photoluminescence of various dye molecules --- p.21 / Chapter 3.4.2 --- Study the two photon luminescence (TPL) of Au NRs --- p.23 / Chapter 3.5 --- UV-Vis-NIR spectrophotometer and fluorescence spectrophotometer --- p.25 / Chapter 3.6 --- Flow-cytometry --- p.26 / Chapter 3.7 --- Western plot --- p.28 / Chapter 3.8 --- Colormetric assays and other biological labels --- p.28 / Chapter 3.8.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test --- p.28 / Chapter 3.8.2 --- Mitochondria, lysosome and nucleus staining --- p.30 / Chapter 3.8.3 --- Detection of apoptosis --- p.31 / Chapter 3.8.4 --- Detection of various reactive oxygen species (ROS) --- p.31 / Chapter Chapter 4 --- Silica NPs interact with human cells --- p.34 / Chapter 4.1 --- Introduction --- p.34 / Chapter 4.2 --- Characterization of silica nanoparticles --- p.36 / Chapter 4.3 --- General description of the NPs’ uptaking and excreting process --- p.42 / Chapter 4.4 --- Tracking of NPs inside the cells --- p.53 / Chapter 4.5 --- Factors influencing the NP-cell interaction and exocytosis process --- p.55 / Chapter 4.5.1 --- The effect of serum (in the incubation medium) on cellular uptake --- p.55 / Chapter 4.5.2 --- Crystallinity effectdistribution of amorphous and crystalline SiO₂ NPs in the cells --- p.57 / Chapter 4.5.3 --- Factors affecting the exocytosis process --- p.59 / Chapter 4.6 --- Cytotoxic effect of silica NPs --- p.60 / Chapter 4.7 --- Conclusion --- p.63 / Chapter Chapter 5 --- Genotoxic effect specifically induced by crystalline SiO₂ nanoparticles in p-53 deficient human cells --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- The difference between crystalline and amorphous silica NPs --- p.66 / Chapter 5.2.1 --- Mitochondria multiplication specially induced by crystalline silica NPs --- p.68 / Chapter 5.2.2 --- DNA fragmentation specially observed in crystalline silica NPs treated cells --- p.71 / Chapter 5.3 --- The cell line sensitive cytotoxicity of crystalline silica NPs --- p.79 / Chapter 5.3.1 --- A general phenomenon of mitochondria increase in p-53 negative cell lines --- p.80 / Chapter 5.3.2 --- General biological consequence of such mitochondria increase --- p.82 / Chapter 5.4 --- Conclusion --- p.83 / Chapter Chapter 6 --- Surface plasmon enhanced drug efficacy for PDT using core shell Au@SiO₂ nanoparticle carrier --- p.84 / Chapter 6.1 --- Introduction --- p.84 / Chapter 6.1.1 --- Brief introduction to the photodynamic therapy (PDT) and photosensitizer (PS) --- p.84 / Chapter 6.1.2 --- Brief introduction to the SPR enhanced generation of ROS --- p.86 / Chapter 6.2 --- Using Au@SiO₂ NPs as drug carrier --- p.88 / Chapter 6.2.1 --- Growth of gold NRs and their controllable oxidation --- p.88 / Chapter 6.2.2 --- Preparation and characterization of Au@(SiO₂-MB) NPs --- p.90 / Chapter 6.2.3 --- Confirmation of MB loading into silica shell --- p.92 / Chapter 6.3 --- Enhanced PDT drug (MB) efficacy when loaded in Au@SiO₂ NPs --- p.95 / Chapter 6.3.1 --- Cellular uptake pathway of free MB and Au@SiO₂ NPs --- p.95 / Chapter 6.3.2 --- Comparing the efficacy of free MB, SiO₂-MB NPs and Au@(SiO₂MB) NPs --- p.98 / Chapter 6.4 --- Studying the behavior of free MB and Au@(SiO₂-MB) NPs as PDT agent --- p.100 / Chapter 6.4.1 --- Comparing the ability of generating ROS by free MB and Au@(SiO₂MB) NPs --- p.100 / Chapter 6.4.2. --- Comparing the types of ROS generated by free MB and Au@(SiO₂MB) NPs --- p.103 / Chapter 6.4.3 --- Comparing the cellular death pathway triggered by free MB and Au@(SiO₂-MB) NPs --- p.105 / Chapter 6.5. --- Discussion on the mechanism for the enhanced efficacy --- p.109 / Chapter 6.5.1 --- Excluding the photothermal effect of Au NRs core --- p.109 / Chapter 6.5.2 --- The role of SiO₂ in the Au@SiO₂ NPs carrier --- p.111 / Chapter 6.5.3 --- Attributing the enhanced efficacy to plasmonic effect of Au NRs core --- p.112 / Chapter 6.6 --- Exploring the potential of using Au@SiO₂ NP carrier in vivo --- p.114 / Chapter 6.7 --- Conclusion --- p.116 / Chapter Chapter 7 --- Conclusion --- p.118 / References --- p.120

ABC運輸蛋白的過度表達是多重抗藥性（MDR）的重要機制之一，癌細胞會同時對結構上無關的抗癌藥物產生抗藥性。避免癌細胞的多重抗藥性有不同方法，其中用聚合物納米載體來攜帶易受多重抗藥性影響的抗癌藥物近年來獲得了很大的關注。本研究的目標在使用一個相對新穎的納米開發技術，被稱為瞬時納米沉澱（FNP），去開發一種運載著易受多重抗藥性影響的抗癌藥物的聚合物納米粒子系統。為此，我們使用專門設計的四流多進旋渦混合器（MIVM），把阿黴素（DOX），一種屬於蒽環類的抗癌藥物，亦同時作為P糖蛋白（P-gp）底物的藥物，包進在二嵌段共聚物內。 / 目的：本研究的目的是:（一）通過MIVM，利用瞬時納米沉澱去配製運載DOX的聚合物納米粒子；（二）辨别和優化納米粒子的大小，物理性能和運載DOX聚合物納米粒子的體外釋放速率；（三）檢查納米粒子的表面元素和化學組成;(四）評估優化納米粒子在抗藥性癌症細胞模型的抗腫瘤能力和抵抗多重抗藥性的能力。 / 方法：不同藥物（DOX)對聚合物比例的瞬時納米沉澱是通過在四流MIVM中混合溶在有機溶液二甲基甲酰胺（DMF)或二甲基酮（ACT)的鹽酸阿黴素(DOX.HC1)或阿黴素游離鹼（DOX.FB)和兩親性二嵌段共聚物[聚乙二醇-聚乳酸；分子量2000-10000]和反抗溶劑（含有氫氧化鈉為DOX.HCl或純淨水DOX+FB)來製備的。納米混懸劑的平均粒徑和粒度分佈會通過動態光散射粒度分析法去檢測，表面電荷會通過界達電位測量去檢測。阿黴素的包封率和載藥量會用紫外/可見光譜儀在波長為480 nm時測定。粒子形態將會用原子力顯微鏡（AFM)來去檢測，粒子表面的組合物將會用X-射線光電子能譜（XPS)來去檢測DOX聚合物納米粒子在不同pH值的的體外釋放會通過紫外/可見光譜儀去檢測。DOX聚合物納米粒子的體外細胞毒性會利用橫若丹明B比色法檢定，藥物積累和反轉運會利用流式細胞儀分析來測定。 / 結果：在適當優化鹽酸阿黴素（DOX.HC1)或阿黴素游離鹼（DOX.FB)的聚合物的比例後，我們成功製備了平均粒徑小於100 nm的DOX聚合物納米粒子(DOX.NP)與使用在有機溶液中DOX.HC1和水相的氫氧化鈉中和法相比，通過在有機溶液中的DOX.FB和純水作為反溶劑來製備的DOX.NP表現出類似的平均粒子大小（小於100 nm)，但顯示出更高的藥物包封率（48 %， 而不是中和法的25 %)。用DOX.FB製備的DOX.NP的載藥量可達14 %DOX.NP表現出pH依賴性的藥物釋放曲線，和在酸性pH值時更强的累積釋放率。X-射線光電子能譜顯示沒有阿黴素出現在納米粒子的表面上P-gp過度表達的LCC6抗藥性乳腺癌细胞的細胞毒性作用顯示了 DOX.NP和DOX.HC1在缓衝溶液中的差異並不顯著。相對DOX.HC1，流式細胞儀分析確定了 DOX.NP明顯增加了細胞攝取DOX的能力。此外，在外排後，DOX.NP在細胞內DOX的濃度顯示出了更長的保留時間。 / 結論：一種通過在多進旋過混合器（MIVM)進行反溶劑沉澱，用於配製具有可控的粒子大小運載DOX的聚合物納米粒子的快速，方便，和可重複性的方法已經被開發。配製的納米粒子顯示出pH值依賴性持續的藥物釋放曲線和更強的癌細胞攝取DOX能力。 / Over-expression of ATP-binding cassette (ABC) is one of the most important mechanisms responsible for multidrug resistance (MDR), in which tumor cells exhibit simultaneous resistance to structurally unrelated anticancer drugs. Various approaches have been attempted to circumvent MDR in cancer cells, among which polymeric nanocarrier for delivery of MDR-sensitive anticancer drugs has received considerable attention in recent years. The present project was aimed at developing a polymeric nanoparticle system using a relatively novel nanoparticle technology termed flash nanoprecipitation (FNP) for delivery of MDR-susceptible chemotherapeutic agents. To this end, doxorubicin (DOX), an anthracycline anticancer agent and a P-gp substrate, was incorporated into an amphiphilic diblock copolymer using a specially designed four-stream multi-inlet vortex mixer (MIVM). / PURPOSES: The objectives of the present study are: (a) to formulate DOX-loaded polymeric nanoparticles by FNP using an MIVM; (b) to characterize and optimize the particle size, physical properties and in vitro DOX release rate of the formulated nanoparticles; (c) to examine the surface elemental and chemical compositions of the formulated nanoparticles; (d) to evaluate the anti-tumor activity of the optimized nanoparticles and their ability to combat MDR in resistant cancer cell line models. / METHODS: FNP of DOX was effected in a four-stream MIVM by mixing organic solutions of doxorubicin hydrochloride (DOX.HCl) or doxorubicin free base (DOX.FB) and an amphiphilic diblock copolymer [polyethylene glycol-polylactic acid (PEG-PLA); MW2k-10 ki]n dimethylformamide (DMF) or acetone (ACT) at different drug-to-polymer ratios with an antisolvent (water containing sodium hydroxide for DOX.HCl or pure water for DOX.FB). The resulting nanosuspensions were characterized for mean particle size and size distribution by dynamic light scattering particle size analysis; surface charges by zeta potential measurements; drug encapsulation efficiency and drug loading by UV/visible spectroscopy at 480 nm; particle morphology by atomic force microscopy (AFM); and surface composition by x-ray photoelectron spectroscopy (XPS). In vitro DOX release from the nanoparticles was measured at different pHs by UV/visible spectroscopy. In vitro cytotoxicity was evaluated by Sulforhodamine B colorimetric assay, and drug accumulation and efflux were determined by flow cytometric analysis. / RESULTS: DOX-loaded polymeric nanoparticles (DOX.NP) with mean particle size below 100 nm were obtained after appropriate optimization of the DOX.HCl or DOX.FB to polymer ratio. Compared with the neutralization method using DOX.HCl in the organic phase and sodium hydroxide in the aqueous phase, DOX.NP prepared with DOX.FB in the organic phase and pure water as antisolvent exhibited a similar mean particle size (< 100 nm) but a significantly higher drug encapsulation efficiency (48% as opposed to 25% for the neutralization method). Drug loading of DOX.NP prepared with DOX.FB could reach up to 14%. DOX.NP exhibited a pH-dependent drug release profile with a much higher cumulative release rate at acidic pHs. XPS revealed that no DOX was present on the nanoparticle surface. The cytotoxic effect on P-gp over-expressing LCC6/MDR cell line revealed insignificant differences between DOX.NP and DOX.HCl in buffered aqueous media. DOX.NP exhibited a marked increase in DOX cellular uptake relative to free DOX, as determined by flow cytometric analysis. Furthermore, DOX.NP showed a significant retention of intracellular concentration of DOX after efflux. / CONCLUSION: A rapid, convenient, and reproducible method for generating DOX-loaded polymeric nanoparticles with controllable particle size through antisolvent precipitation in a multi-inlet vortex mixer has been developed. The formulated nanoparticles displayed a pH-dependent sustained drug release profile and an enhanced DOX uptake into cancer cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tam, Yu Tong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 119-130). / Abstracts also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xiii / ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- Introduction --- p.1 / Chapter 1.1 --- Rationale of the Study --- p.2 / Chapter 1.2 --- Doxorubicin --- p.3 / Chapter 1.2.1 --- Origin --- p.3 / Chapter 1.2.2 --- Physico-chemical properties --- p.6 / Chapter 1.2.3 --- Mechanism of Action --- p.7 / Chapter 1.2.4 --- Multidrug Resistance in Cancer --- p.7 / Chapter 1.2.4.1 --- Mechanisms of Multidrug Resistance --- p.8 / Chapter 1.3 --- Nanoparticles for Cancer Therapy --- p.9 / Chapter 1.3.1 --- Properties of Nanoparticles --- p.9 / Chapter 1.3.1.1 --- Small Particle Size --- p.10 / Chapter 1.3.1.2 --- High Payload Density --- p.11 / Chapter 1.3.1.3 --- Flexible Modification of Surface Properties --- p.11 / Chapter 1.3.2 --- Targeted Cancer Therapy --- p.12 / Chapter 1.3.2.1 --- Passive Tumor Targeting --- p.13 / Chapter 1.3.2.2 --- Active Tumor Targeting --- p.14 / Chapter 1.3.3 --- Reversal of Multidrug Resistance --- p.15 / Chapter 1.3.3.1 --- Endocytosis of Nanoparticles --- p.16 / Chapter 1.3.4 --- Nanoparticle Approaches to Anti-cancer Drug Delivery --- p.17 / Chapter 1.3.4.1 --- Liposomes --- p.18 / Chapter 1.3.4.2 --- Polymeric Nanoparticles --- p.18 / Chapter 1.4 --- Fabrication of Nanoparticles --- p.19 / Chapter 1.5 --- Aims and Scope of the Present Study --- p.21 / Chapter CHAPTER 2. --- Materials & Methods --- p.23 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Chemicals --- p.24 / Chapter 2.1.2 --- Materials for Cell Culture --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Preparation of Doxorubicin Nanoparticles by Flash Nanoprecipitation --- p.26 / Chapter 2.2.1.1 --- Acid-Base Neutralization during Mixing --- p.26 / Chapter 2.2.1.2 --- Preparation of Doxorubicin Free Base before Mixing --- p.29 / Chapter 2.2.1.2.1 --- Doxorubicin Free Base Preparation --- p.29 / Chapter 2.2.2 --- Determination of Particle Size and Zeta Potential --- p.30 / Chapter 2.2.3 --- Co-stabilizers and Particle Stability --- p.30 / Chapter 2.2.4 --- Chemical Stability of Doxorubicin --- p.31 / Chapter 2.2.5 --- Determination of Encapsulation Efficiency --- p.31 / Chapter 2.2.5.1 --- Calibration Curve of Doxorubicin --- p.33 / Chapter 2.2.5.2 --- Dialysis --- p.33 / Chapter 2.2.5.3 --- Ultrafiltration --- p.35 / Chapter 2.2.6 --- Determination of Drug Loading --- p.35 / Chapter 2.2.6.1 --- Freeze Drying --- p.36 / Chapter 2.2.7 --- Morphological Examination --- p.36 / Chapter 2.2.7.1 --- X-ray Photoelectron Spectroscopy --- p.36 / Chapter 2.2.7.2 --- Atomic Force Microscopy --- p.36 / Chapter 2.2.8 --- In vitro release study --- p.37 / Chapter 2.2.8.1 --- Experimental Protocols --- p.37 / Chapter 2.2.8.2 --- Calculation of Cumulative Drug Release --- p.37 / Chapter 2.2.9 --- In vitro cytotoxicity study --- p.38 / Chapter 2.2.9.1 --- Sulforhodamine B Colorimetric Assay --- p.38 / Chapter 2.2.10 --- Cellular Uptake study --- p.39 / Chapter 2.2.10.1 --- Drug Accumulation Assay --- p.39 / Chapter 2.2.10.1 --- Drug Efflux Assay --- p.39 / Chapter 2.2.11 --- Analytical techniques --- p.40 / Chapter 2.2.11.1 --- UV/Vis Analysis --- p.40 / Chapter 2.2.11.2 --- HPLC Analysis --- p.40 / Chapter 2.2.12 --- Statistical analysis --- p.41 / Chapter CHAPTER 3. --- Results & Discussions --- p.42 / Chapter 3.1 --- Preparation of Doxorubicin Nanoparticles by Flash Nanoprecipitation --- p.43 / Chapter 3.1.1 --- Acid-Base Neutralization during Mixing --- p.44 / Chapter 3.1.1.1 --- Influence of Drug Concentration --- p.44 / Chapter 3.1.1.2 --- Influence of Alkaline Medium --- p.48 / Chapter 3.1.1.3 --- Influence of Drug-to-Polymer Ratios --- p.53 / Chapter 3.1.1.4 --- Particle Stability --- p.54 / Chapter 3.1.1.5 --- Co-stabilizers Tests on Stability --- p.55 / Chapter 3.1.1.5.1 --- Effect of PEG-PLA Co-polymers --- p.55 / Chapter 3.1.1.5.2 --- Effect of Co-stabilizers --- p.56 / Chapter 3.1.2 --- Preparation of Doxorubicin Free Base before Mixing --- p.62 / Chapter 3.1.2.1 --- Influence of Solvent System --- p.62 / Chapter 3.1.2.2 --- Influence of Drug-to-Polymer Ratios --- p.65 / Chapter 3.1.2.3 --- Drug Loading --- p.65 / Chapter 3.1.2.4 --- Particle Stability --- p.68 / Chapter 3.1.2.4.1 --- Concentrated Particle Stability --- p.73 / Chapter 3.2 --- Stability Studies on Doxorubicin Nanoparticle at Physiological and Cancer Cell pHs --- p.75 / Chapter 3.2.1 --- Chemical Stability --- p.75 / Chapter 3.2.2 --- Physical Stability --- p.77 / Chapter 3.3 --- In vitro Release Study --- p.79 / Chapter 3.4 --- Morphological Examination --- p.86 / Chapter 3.4.1 --- Zeta Potential --- p.92 / Chapter 3.5 --- In vitro Cellular Study --- p.93 / Chapter 3.5.1 --- Cellular Uptake Study --- p.93 / Chapter 3.5.1.1 --- Drug Accumulation and Drug Efflux --- p.93 / Chapter 3.5.2 --- Cytotoxicity of Blank Nanoparticles --- p.98 / Chapter 3.5.3 --- Cytotoxicity of DOX loaded Nanoparticles --- p.100 / Chapter CHAPTER 4. --- Conclusions --- p.106 / APPENDIX --- p.109 / REFERENCES --- p.118

The advance of nanotechnology has made it possible that pure silver can be engineered into nano scale level with less than 100 nm in size. So far many studies have confirmed anti-bacterial and anti-inflammatory efficacy of silver nanoparticles (AgNPs). In our previous study we have revealed that AgNPs could promote wound healing through modulation of cytokines in a burn wound model in mice. Nonetheless, the exact effects mediated by AgNPs on various cell types in skin, including keratinocytes and fibroblasts, during wound healing still remain unknown. Therefore, in the present study we targeted a full-thickness excisional wound model in mice to explore the action and potential toxicity of AgNPs on keratinocytes and fibroblasts. Immunohistochemistry staining and molecular assay were conducted to explore AgNPs-induced re-epithelization and cell differentiation in both in vivo and in vitro studies. We next targeted the healed skin after AgNPs-mediated wound healing using tensile test to compare their mechanical function. Meanwhile, immunohistochemistry staining and quantitative assay were utilized to further investigate and compare collagen deposition, and scanning electron microscopy (SEM) was used to observe the morphology and distribution of collagen fibrils in healed skin. Moreover, AgNPs of different sizes and doses were studied to investigate the potential toxicity, their influence on cell migration, and extracellular matrix (ECM) production. Key results: 1. AgNPs could accelerate excisional wound healing in mouse skin when compared with other formats of silver. 2. AgNPs mediated differential cellular response in skin cells. They promoted proliferation and migration of keratinocytes in epidermal layer, through which the re-epithelization process during wound healing was enhanced; while proliferation of fibroblasts in dermal layer was inhibited and they were driven into the differentiation of myofibroblasts, through which wound contraction process was strengthened. 3. AgNPs could suppress the proliferation of human keloid fibroblasts and ECM production including collagen, fibronectin and heat shock protein, which would suggest that AgNPs had anti-fibrosis effect. 4. The AgNPs could stimulate the proliferation of epidermal progenitors and their differentiation into keratinocytes during wound healing. This biological event further contributed to the re-epithelization process. 5. AgNPs-mediated healed skin possessed comparable mechanical function, collagen deposition and fibril alignment to normal skin, which suggested AgNPs could modulate collagen production during skin wound healing. 6. The inhibitory effect on fibroblasts and cytotoxicity mediated by AgNPs showed a dose-dependent and size-dependent manner. In conclusion, AgNPs not only contribute to healing of infected skin wounds through antibacterial activity, but can also accelerate wound healing through mediating differential cellular responses in different skin cell types and modulate collagen production during wound healing. Furthermore, there should be an optimal concentration and size to exert maximal biological action with minimal toxicity for each specific cell type. Present studies further extended our knowledge of AgNPs and have implications for treatment of wounds in clinical setting. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy