Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Nos últimos anos têm crescido cada vez mais o número de pesquisas envolvendo nanotecnologia para obtenção de medicamentos com liberação controlada, pois esses sistemas podem: proteger o fármaco de incompatibilidades tanto biológicas quanto físico-químicas assim como controlar a biodisponibilidade do fármaco. Embora com todas essas vantagens não existem métodos in vitro realmente capazes de prever com precisão a liberação dos fármacos por esses sistemas, por esse motivo, é muito importante o desenvolvimento de métodos de liberação in vitro para determinar a cinética de liberação desses sistemas.O presente trabalho teve como objetivo desenvolver e validar os métodos de eletroforese capilar (CE) e cromatografia líquida de alta eficiência (HPLC) para determinar a eficiência de encapsulação do fármaco imatinibe em nanopartículaspreviamente elaboradas e caracterizadas, assim como estudar sua liberação in vitro por CE. As nanopartículas foramdesenvolvidas pelo método de nanoprecipitaçãoe caracterizadas quanto ao tamanho, potencial zeta, morfologia e eficiência de encapsulação. A eletroforese capilar é uma técnica alternativa muito promissora em relação ao HPLC devido ao seu baixo custo, menor tempo de corrida e menos poluente ao meio ambiente. Os métodos de quantificação por CE e HPLCforam desenvolvidose validadossegundo as diretrizes do ICH, Farmacopeia Americana e ANVISA, permitindo desenvolver um estudo de liberação.As nanoesferas desenvolvidas apresentaram diâmetro médio próximo a 150nm, com índice de polidispersão menor que 0,1 e aproximadamente 90% de eficiência de encapsulação. Ambos métodos se mostraram lineares com coeficientes de determinação superiores a 0,99, os métodos se mostraram precisos (%DPR< 2), exatos(101,0±4,2% e 98,0±2,5% para HPLC e CE, respectivamente)e seletivos.O método de CE permitiu desenvolver um método de estudo de liberação independente das membranas de diálise. / In recent years, there has been a growing number of researches involving nanotechnology to obtain controlled release drugs, these systems can: protect the drug against biological and physico-chemical incompatibilities; controlling the bioavailability of the drug. Although with all these advantages there are no in vitro methods really capable of accurately predicting drugs release by such systems, therefore, the development of in vitro release methods to determine the release kinetics of such systems is very important. The objective of the present work was to develop and validate capillary electrophoresis (CE) and HPLC methods to determine the encapsulation efficiency of the imatinib drug in previously elaborated and characterized nanoparticles, as well as to study its release in vitro by CE method. The nanoparticles were synthesized using the nanoprecipitation method and characterized by size, zeta potential, morphology and encapsulation efficiency. Capillary electrophoresis is a very promising alternative to HPLC because of its low cost, less runtime and less polluting environment. The CE and HPLC methodswere developed and validated according ICH, American Pharmacopoeia and ANVISA guidelines.Developed nanospheres had an average diameter close to 150nm, with polydispersity index less than 0.1 and approximately 90% encapsulation efficiency. Both methods were linear with determination coefficients higher than 0.99, the methods were precise (%RSD < 2), accurate (101.0±4,2% and 98.0±2,5% for HPLC and CE, respectively) and selective. Capillary electrophoresis method allowed to develop a drug release study independent of dialysis membranes.

by Lo Hoi Ka Elka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / LIST OF TABLES AND FIGURES --- p.viii / ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1 : --- INTRODUCTION / Chapter 1.1. --- DIFFERENT TREATMENTS OF THE CANCER THERAPY --- p.1 / Chapter 1.2. --- THE SIDE EFFECTS OF CANCER TREATMENT / Chapter 1.2.1. --- Surgery --- p.1 / Chapter 1.2.2. --- Radiotherapy --- p.2 / Chapter 1.2.3. --- Chemotherapy --- p.2 / Chapter 1.3. --- THE CHARACTERISTICS OF DOXORUBICIN (DOX) / Chapter 1.3.1. --- The structure of Dox --- p.6 / Chapter 1.3.2. --- The actions of Dox --- p.8 / Chapter 1.3.3. --- The adverse side effect of Dox --- p.8 / Chapter 1.4. --- THE RATIONALE OF USING LOW DENSITY LIPOPROTEIN (LDL) AS A TARGET CARRIER IN CANCER THERAPY / Chapter 1.4.1. --- The correlation between cholesterol and cancer --- p.9 / Chapter 1.4.2. --- Low density lipoprotein (LDL) as a target carrier --- p.11 / Chapter 1.4.3. --- The down and up regulation of LDL receptors --- p.14 / Chapter 1.4.4. --- The characteristics of Fuctus Craegus (FC) --- p.15 / Chapter 1.5. --- DIFFERENT METHODS OF THE PREPARATION OF THE LOW DENSITY LIPOPROTEIN-DRUG (LDL- DRUG) --- p.18 / Chapter 1.6. --- THE CHARACTERISTICS OF LOW DENSITY LIPOPROTEIN (LDL) / Chapter 1.6.1. --- The structure of LDL --- p.20 / Chapter 1.6.2. --- The metabolic pathway of LDL in human bodies --- p.23 / Chapter 1.7. --- THE MULTIDRUGS RESISTANCE IN TUMOR CELLS --- p.25 / Chapter 1.7.1. --- The mechanism of multidrug resistance --- p.27 / Chapter 1.7.2. --- The structure of P-glycoprotein --- p.27 / Chapter 1.7.3. --- The mechanism of P-glycoprotein --- p.30 / Chapter 1.8. --- COMBINED TREATMENT WITH HYPERTHERMIA --- p.31 / Chapter 1.9. --- AIM OF THE STUDY --- p.33 / Chapter CHAPTER 2 : --- MATERIALS AND METHODS / Chapter 2.1. --- MATERIALS / Chapter 2.1.1. --- Animals --- p.34 / Chapter 2.1.2. --- Buffers --- p.34 / Chapter 2.1.3. --- Cell culture reagents --- p.36 / Chapter 2.1.4. --- Chemicals --- p.38 / Chapter 2.1.5. --- Culture of cells --- p.40 / Chapter 2.2. --- METHODS / Chapter 2.2.1. --- In vitro studies / Chapter 2.2.1.1. --- "LDL, doxorubicin complex formation" --- p.41 / Chapter 2.2.1.2. --- Determination of the concentration of LDL-Dox --- p.42 / Chapter 2.2.1.3. --- In vitro cytotoxicity --- p.43 / Chapter 2.2.1.4. --- The cytotoxicity of the combined treatment with anticancer drugs --- p.44 / Chapter 2.2.1.5. --- The preparation of Fructus Crataegus (FC) --- p.46 / Chapter 2.2.1.6. --- Western blot --- p.47 / Chapter 2.2.1.7. --- Flow cytometry --- p.49 / Chapter 2.2.1.8. --- Confocal laser scanning microscopy --- p.52 / Chapter 2.2.2. --- In vivo studies / Chapter 2.2.2.1. --- Subcutaneous injection of R-HepG2 cells in nude mouse --- p.55 / Chapter 2.2.2.2. --- Treatment schedules --- p.55 / Chapter 2.2.2.3. --- Assay of investigating of the myocardial injury --- p.56 / Chapter 2.2.2.4. --- Tissue preparation procedure for light microscope (LM) --- p.57 / Chapter 2.2.3. --- Statistical analysis in our research --- p.59 / Chapter CHAPTER 3 : --- RESULTS / Chapter 3.1. --- in vitro STUDIES / Chapter 3.1.1. --- The preparation of low density lipoprotein-doxorubicin (LDL-Dox) --- p.60 / Chapter 3.1.2. --- Studies on human hepatoma cells line (HepG2 cells) / Chapter 3.1.2.1. --- The comparison of Dox and LDL-Dox accumulated in HepG2 cells --- p.63 / Chapter 3.1.2.2. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in HepG2 cells --- p.65 / Chapter 3.1.2.3. --- The comparsion of the cytotoxicity of Dox and LDL-Dox on HepG2 cells --- p.67 / Chapter 3.1.2.4. --- The comparison of the cytotoxicty of Dox and LDL-Dox with and without hyperthermia on HepG2 cells --- p.73 / Chapter 3.1.2.5. --- The comparison of accumulation of Dox and LDL-Dox in HepG2 cells treated with and without combination of with hyperthermia --- p.77 / Chapter 3.1.2.6. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in HepG2 treated cells with and without hyperthermia --- p.80 / Chapter 3.1.2.7. --- Modulation of LDL receptors on HepG2 cells------Up- regulation of LDL receptors by Fructus Craegtus (FC) / Chapter 3.1.2.7.1. --- The comparsion of LDL receptor expression on HepG2 cells after Fructus Craegtus (FC) pre-treatment --- p.83 / Chapter 3.1.2.7.2. --- The comparison of accumulation of LDL-Dox accumulated in HepG2 cells pre-treated with and without Fructus Craegtus (FC) --- p.85 / Chapter 3.1.2.7.3. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of LDL-Doxin HepG2 cells after Fructus Craegtus (FC) pre- treatment --- p.88 / Chapter 3.1.2.7.4. --- Cytotoxicity of combined treatment with LDL-Dox and Fructus Craegtus (FC) --- p.91 / Chapter 3.1.3. --- Studies on multidrug human resistant hepatoma cell line (R-HepG2 cells) / Chapter 3.1.3.1. --- The overexpression level of P-glycoprotein in resistant cell line R-HepG2 --- p.93 / Chapter 3.1.3.2. --- The comparison of Dox and LDL-Dox accumulated in R- HepG2 cells --- p.95 / Chapter 3.1.3.3. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in R-HepG2 cells --- p.97 / Chapter 3.1.3.4. --- The comparsion of the cytotoxicity of Dox and LDL-Dox on R-HepG2 cells --- p.99 / Chapter 3.1.3.5. --- The comparison of the cytotoxicty of Dox and LDL-Dox with and without hyperthermia on R-HepG2 cells --- p.109 / Chapter 3.1.3.6. --- The comparison of the accumulation of Dox and LDL- Dox in R-HepG2 cells treated in combination with hyperthermia --- p.113 / Chapter 3.1.3.7. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in R-HepG2 cells with and without hyperthermia --- p.117 / Chapter 3.1.3.8. --- Modulation of LDL receptors on R-HepG2 cells ------ Up-regulation of LDL receptors by Fructus Craegtus (FC) / Chapter 3.1.3.8.1. --- The comparsion of LDL receptor expression on R-HepG2 cells after Fructus Craegtus (FC) pre-treatment --- p.120 / Chapter 3.1.3.8.2. --- The comparsion of the accumulation of LDL- Dox in R-HepG2 cells after Fructus Craegtus (FC) pre-treatment --- p.122 / Chapter 3.1.3.8.3. --- Confocal laser scanning microscopic (CLSM) studies in the accumulation of LDL-Dox by Fructus Craegtus pre-treatment in R-HepG2 cells --- p.125 / Chapter 3.1.3.8.4. --- The comparison of cytotoxicity of combined treatment with LDL-Dox and Fructus Craegtus (FC) in R-HepG2 cells --- p.128 / Chapter 3.2. --- in vivo STUDIES / Chapter 3.2.1. --- The comparison of Dox and LDL-Dox on reducing the tumor sizes and weight in nude mice bearing R-HepG2 cells / Chapter 3.2.1.1. --- The comparison of Dox and LDL-Dox on reducing the tumor size in nude mice bearing R-HepG2 cells --- p.130 / Chapter 3.2.1.2. --- The comparison of Dox and LDL-Dox on reducing the tumor weight in nude mice bearing R-HepG2 cells --- p.138 / Chapter 3.2.2. --- Myocardial injury measured by Lactate dehydrogenase (LDH) activity in nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox --- p.140 / Chapter 3.2.3. --- Myocardial injury measured by Creatine kinase (CK) activity in nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox --- p.143 / Chapter 3.2.4. --- Histological studies of heart of nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox / Chapter 3.2.4.1. --- Heart section of nude mice --- p.146 / Chapter 3.2.4.2. --- Heart section of nude mice bearing R-HepG2 cells --- p.148 / Chapter 3.2.4.3. --- Heart section of lmg/kg Dox treated nude mice bearing R- HepG2 cells --- p.150 / Chapter 3.2.4.4. --- Heart section of 2mg/kg Dox treated nude mice bearing R- HepG2 cells --- p.152 / Chapter 3.2.4.5. --- Heart section of lmg/kg LDL-Dox treated nude mice bearing R-HepG2 cells --- p.154 / Chapter CHAPTER 4 --- : DISCUSSION / Chapter 4.1. --- in vitro STUDIES / Chapter 4.1.1. --- The cytotoxicity of Dox and LDL-Dox on HepG2 cells and R- HepG2 cells --- p.156 / Chapter 4.1.2. --- The combined treatment on HepG2 cells and R-HepG2 cells --- p.157 / Chapter 4.1.3. --- The modulation of LDL-R expression --- p.159 / Chapter 4.2. --- in vivo STUDIES --- p.162 / Chapter CHAPTER 5 --- : CONCLUSION / Chapter 5.1. --- CONCLUSION / Chapter 5.1.1. --- In vitro studies --- p.167 / Chapter 5.1.2. --- In vivo studies --- p.169 / Chapter 5.2. --- FUTURE PROSPECTIVE --- p.170 / REFERENCES --- p.172

Lipossomas (LUV) são estruturas compostas por uma bicamada lipídica que se organizam de forma semelhante a vesículas, contendo um compartimento aquoso em seu interior. Têm sido avaliados como potenciais carreadores de fármacos. No entanto, após sua administração, in vivo, opsoninas do soro adsorvem-se em sua superfície contribuindo para que o sistema fagocitário mononuclear (SFM) reconheça essas partículas, favorecendo sua remoção da circulação. O sistema complemento (SC) parece ter papel importante neste processo, principalmente por gerar fragmentos ativos do componente C3 (C3b/iC3b) que se depositam nas vesículas lipossomais e são reconhecidos por receptores do complemento presentes, por exemplo, nos polimorfonucleares. Antioxidantes, como a quercetina, têm demonstrado importantes e benéficos efeitos sobre a saúde humana, porém sua baixa solubilidade em água e biodisponibilidade limitam seu uso. Assim, o desenvolvimento apropriado de carreadores de flavonoides seria de grande importância para sua aplicabilidade in vivo. O objetivo do presente trabalho é avaliar a ativação das proteínas do SC por lipossomas compostos de fosfatidilcolina de soja e colesterol (PC:CHOL) ou colesteril-etil-éter (PC:CHOL-OET), contendo ou não quercetina. O consumo das vias clássica (VC) e alternativa (VA) provocado pelas diferentes vesículas foi analisado por ensaio hemolítico e a quantificação de iC3b e anticorpos naturais (IgG e IgM) na superfície dessas partículas foi realizada através de kits de ELISA. A ativação de C3 por vesículas contendo ou não quercetina foi avaliada por imunoeletroforese bidimensional (IEF). Os resultados mostram que lipossomas vazios, compostos por grande quantidade de colesterol, consomem mais os componentes do complemento para ambas as vias, VC e VA. Ainda, a substituição de colesterol por colesteril-etil éter reduziu o consumo das duas vias, mas a ativação do SC ainda é dependente da quantidade deste composto. A incorporação de quercetina não alterou o consumo de ambas as vias. O depósito de iC3b, IgG ou IgM nas vesículas compostas de PC:CHOL-OET na proporção de massa 3:1 foi o menor comparado aos demais. A IEF mostrou que vesículas PC:CHOL vazias induzem maior clivagem de C3 em relação às vesículas PC:CHOL-OET. Ainda, a incorporação de quercetina reduz a conversão de C3 em seus fragmentos. Essas observações sugerem que a preparação lipossomal PC:CHOL-OET em proporção de massa 3:1 parece ser a mais adequada para dar continuidade aos estudos que deverão culminar na tentativa de utilizá-la como carreadora de quercetina para administração in vivo / Liposomes (LUV) are structures composed by lipid bilayer that are organized similarly to vesicles, containing an aqueous compartment inside. They have been evaluated as potential drug carriers, however, after in vivo administration, serum opsonins are adsorb on the surface, contributing to their clearance from the circulation by mononuclear phagocytes system (MPS). The complement system (CS) seems to play an important role in this process, mainly to generate active fragments of the C3 component (C3b/iC3b) that are deposited in the liposomal vesicles and are recognized by complement receptors present, for example, in polymorphonuclear cells. Antioxidants such as quercetin have demonstrated significant and beneficial effects on human health, but its low water solubility and bioavailability limit their use. Thus, the proper development of flavonoids carriers would be of great importance to its applicability in vivo. The objective of this study is to evaluate the activation of SC proteins by liposomes composed of soy phosphatidylcholine and cholesterol (PC: CHOL) or cholesteryl ethyl ether (PC: CHOL-OET), with or without quercetin. The consumption of the classical (CP) and alternative pathway (AP) caused by the different vesicles was analyzed by hemolytic assay and quantification of iC3b and natural antibodies (IgG and IgM) on the surface of these particles was performed using ELISA kits. The activation of C3 by vesicles with or without quercetin was assessed by two-dimensional immunoelectrophoresis (IEF). The results show that empty liposomes, composed of large amounts of cholesterol, consume more CS components in both pathways, CP and AP. Moreover the replacement of cholesterol by cholesteryl ethyl ether reduced the consumption of both pathways, but the activation of the SC is still dependent on the amount of the compound. The incorporation of quercetin did not alter the consumption of both pathways. The deposition of iC3b, IgG or IgM in vesicles composed of PC: CHOL-OET at mass ratio of 3:1 was the lowest compared to the others. The IEF showed that empty vesicles of PC:CHOL induce less cleavage of C3 in relation to vesicles of PC: CHOL-OET. In addition, the incorporation of quercetin reduces the conversion of C3 into its fragments. These observation suggest that the liposomes PC:CHOL at mass ratio 3:1 seems to be the most appropriate to continue the studies that could culminate in an attempt to use it as a carrier to administrate quercetin in vivo

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

Stimulus-responsive colloidosomes which completely dissolve upon a mild pH change are developed. pH-Responsive nanoparticles that dissolve upon a mild pH increase are synthesized by a nanoprecipitation method and are used as stabilizers for a double water-in-oil-in-water Pickering emulsion. These emulsions serve as templates for the production of pH-responsive colloidosomes. Removal of the middle oil phase produces water-core colloidosomes that have a shell made of pH-responsive nanoparticles, which rapidly dissolve above pH 7. The permeability of these capsules is assessed by FRAP, whereby the diffusion of a fluorescent tracer through the capsule shell is monitored. Three methods for tuning the permeability of the pH-responsive colloidosomes were developed: ethanol consolidation, layer-by-layer assembly and the generation of PLGA-pH-responsive nanoparticle hybrid colloidosomes. The resulting colloidosomes have different responses to the pH stimulus, as well as different pre-release permeability values. Additionally, fundamental studies regarding the role of particle surface roughness on Pickering emulsification are also shown. The pH-responsive nanoparticles were used as a coating for larger silica particles, producing rough raspberry-like particles. Partial dissolution of the nanoparticle coating allows tuning of the substrate surface roughness while retaining the same surface chemistry. The results obtained show that surface roughness increases the emulsion stability of decane-water systems (to almost twice), but only up to a certain point, where extremely rough particles produced less stable emulsions presumably due to a Cassie-Baxter wetting regime. Additionally, in an octanol-water system, surface roughness was shown to affect the type of emulsion generated. These results are of exceptional importance since they are the first controlled experimental evidence regarding the role of particle surface roughness on Pickering emulsification, thus clarifying some conflicting ideas that exist regarding this issue.

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Edith Mathiowitz. Includes bibliographical references.

The Bio J-FIL process has been demonstrated to be a viable method for manufacturing nanoscale, polymeric drug carriers. The process allows for precise control of the size and shape of the drug carriers. While the original process is sufficient for research scale projects, reliability issues have prevented it from being scalable to levels that could potentially be used for mass-production of the drug carriers. In this thesis, a detailed root cause analysis has been conducted to determine the cause of the reliability issues limiting the Bio JFIL process. A series of experiments with varying substrate and imprint fluid combinations were conducted to pinpoint the cause of imprint failure in the Bio J-FIL process. Upon determining the cause of failure, an alternative imprint process was investigated that sought to increase the variety of materials used in the process by utilizing an intermediary layer. This process is referred to in this thesis as the Bio JFIL-I process. The results using Bio JFIL-I indicated increased reliability over the standard Bio J-FIL process. Further refinement of the Bio JFIL-I process could also address additional issues with the Bio J-FIL process unrelated to process reliability. The Bio JFIL-I approach presented in this thesis is complementary to other approaches that have been recently pursued in the literature which are discussed in the thesis. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [95]-100).