Microgels, colloidal networks of crosslinked water-soluble polymers with dimensions < 1 μm, have been demonstrated to be useful materials in a wide range of biomedical and environmental applications. In particular, temperature-responsive microgels based on poly(N- isopropylacrylamide) (PNIPAM) have attracted significant research interest in drug delivery applications. However, conventional precipitation-based PNIPAM microgels are functionally non-degradable, problematic for biomedical applications. To resolve this issue, a thermally- driven self-assembly approach based on hydrazide and aldehyde functionalized PNIPAM oligomers to form an acid-labile hydrazone bond was developed in the Hoare Lab to produce thermoresponsive, colloidally stable, monodisperse and degradable microgels. In this thesis, the internal structure of these self-assembled microgels was investigated using small and ultra-small angle neutron scattering and surface force experiments. Contrary to expectations based on the assembly technique, all these characterization strategies suggested that self-assembled microgels have a homogeneously cross-linked internal structure. It is anticipated that these well-defined degradable and homogeneous nanoscale gel networks offer opportunities for addressing challenges in drug delivery, biosensing, and optics by exploiting the predictable diffusive and refractive properties of the homogeneous microgel networks. In addition, the co-self-assembly of a moderately hydrophobic anti-inflammatory drug (dexamethasone) during the microgel self-assembly process was demonstrated to enable five-fold higher drug encapsulation (75-80%) relative to the conventional partition/diffusion- based drug loading processes. This result addresses a key challenge in delivering hydrophobic drugs using conventional precipitation-based microgel systems due to the inherent hydrophilicity of the crosslinked network. The potential of the self-assembly approach to fabricate multi-responsive smart microgels was demonstrated by incorporating pH-ionizable functional groups (via the copolymerization of acrylic acid and 2-dimethylaminoethylmethacrylate to introduce anionic and cationic charges respectively) into the hydrazide and aldehyde-functionalized precursor polymers prior to self-assembly. The self-assembled charged microgels showed the same pH- responsive swelling behaviours of conventional microgels, including amphoteric microgels that can be formed at any desired cationic:anionic charge density by simply mixing different ratios of cationic and anionic precursor polymers. Such microgels offer significant potential to improve the performance of microgels in applications demanding dual pH/temperature specific drug delivery. / Thesis / Master of Applied Science (MASc) / Medications can exist in many different forms. From pills to injections, existing drug delivery systems require a high frequency of drug administration and often result in low efficacy of drug once administered to the human body. Polymer-based drug delivery systems have the potential to improve this delivery. In particular, microgels, water-filled crosslinked polymer networks with a size less than one micron, offer promise as a drug delivery vehicle. The size and chemical composition of microgels can be tailored to enable their use in a wide array of drug delivery applications. In addition, microgels can be loaded with a therapeutic agent and transported in the blood stream to deliver drug at a rate and/or location tunable based on the internal structure of the microgel. “Smart” microgels have the particularly attractive ability to change their properties in response to certain environmental stimuli (i.e. temperature or pH). However, current smart microgel systems are non-degradable and would accumulate in the body, causing undesired side-effects. In this thesis, a new self-assembly approach has been used to produce degradable microgels with the potential to switch properties in response to both temperature and pH. Water-insoluble drugs can be encapsulated more efficiently with this method, and the dual-responsive behaviour is expected to improve our capacity to deliver drug at the rate and location desired in the body.

Macromolecular magnetic resonance imaging (MRI) contrast agents have unique localization and contrast enhancement properties. We have designed and studied a monodisperse paramagnetic β-cyclodextrin click cluster (Gd10) decorated with Gd-containing arms and unique contrast enhancing polymers. To synthesize Gd10, a novel alkyne-functionalized diethylenetriaminetetraacetic acid chelate was created and coupled to a per-azido-β-cyclodextrin core and chelated with Gd(III) to yield the precursor macromolecule. Luminescence measurements were carried out using an analogous structure Eu(III)-containing structure and indicated that each lanthanide has an average of 1.8 water exchange sites. Gd10 yields a high relaxivity profile (6.2 mM⁻¹ s⁻¹ per Gd(III) at 9.4 T). Gd10 shows toxicity higher than clinically used contrast agents such as Magnevist&trade in vitro in cardiomyoblast cells. No acute toxicity was observed in the rats (n = 9) and contrast enhanced image analysis indicates renal processes may be involved in clearance. The contrast enhancing polymers we developed are new macromolecular beacons that allow the delivery of nucleic acids to be visualized at different biological scales. They contain repeated oligoethyleneamines, for binding and compacting nucleic acids into nanoparticles, and Gd(III)/Eu(III) chelates. The chelated lanthanides allow the visualization of the delivery vehicle via microscopy and via magnetic resonance imaging (MRI). We demonstrate that these new delivery beacons effectively bind plasmid DNA(pDNA) and protect their cargo nucleic acids from nuclease damage. The lanthanide-chelate materials have been found to efficiently deliver pDNA into cultured cells and do not exhibit toxicity. Micrographs of cultured cells exposed to the nanoparticle complexes formed with fluorescein-labeled pDNA and the europium-chelated polymers reveal effective intracellular imaging of the delivery process. MRI of bulk cells exposed to the complexes formulated with pDNA and the gadolinium-chelated structures show bright image contrast, allowing visualization of effective intracellular delivery on the tissue-scale. Because of their versatility as imaging probes, these delivery beacons posses remarkable potential for tracking and understanding nucleic acid transfer in vitro and have promise for in vivo imaging applications. In later studies the Ln-chelating polymers were co-polymerized with dimethylgalacterate which definitively increases luciferase gene expression (up 50x enhancement) and cellular uptake (up to 2x enhancement). / Ph. D.

Doxorubicin is one of the most widely used and effective anticancer agents to treat a wide spectrum of tumors. But its success in cancer therapy is greatly compromised by its cumulative dose-dependent side effects of cardiotoxicity and tumor cell resistance. For the purpose of addressing these side effects, a gold nanoparticles-based anticancer drug delivery system was designed. Five novel thiolated doxorubicin analogs were designed and synthesized and their biological activities have been evaluated. These doxorubicin analogs and the poly(ethylene glycol) (PEG) stabilizing ligands were conjugated to gold nanoparticles via formation of a gold-thiol bond. The systems were evaluated in vitro and in vivo, and the results show that controlled drug release can be achieved either by acidic conditions or by reducing agents in cancer cells, depending on the design of the thiolated drug construct. The overall drug delivery system should achieve enhanced drug accumulation and retention in cancer cells and favorable drug release kinetics, and should demonstrate therapeutic potential and the ability to address some of the current problems of doxorubicin in cancer therapy. / Master of Science

Pullulan is an exopolysaccharide secreted extracellularly by the black yeast-like fungi Aureobasidium pullulans. Due to an alpha-(1-->6) linked maltotriose repeat unit, which interferes with hydrogen bonding and crystallization, pullulan is completely water soluble unlike cellulose. It has also been tested and shown to possess non-toxic, biodegradable, non-mutagenic and non-carcinogenic properties. Chemical modification of polysaccharides to increased hydrophobicity and increase functionality has shown great promise in drug delivery systems. Particularly in amorphous solid dispersion (ASD) formulations, hydrophobicity increases miscibility with hydrophobic, crystalline drugs and carboxy functionality provides stabilization with drug moieties and well as pH specific release. Successful synthesis of cellulose w-carboxyalkanoates have been reported and showed great promise as ASD polymers based on their ability to retard the recrystallization of the HIV drug ritonavir and antibacterial clarithromycin. However, these cellulose derivatives have limitations due to their limited water solubility. Natural pullulan is water-soluble and modification with w-carboxyalkanoate groups would provide a unique set of derivatives with increased solubility therefore stronger polymer-drug interactions in solution. We have successfully prepared novel pullulan w-carboxyalkanoates, which exhibit good solubility in polar aprotic and polar protic solvents. All derivatives exhibit high thermal stability and most recorded high glass transition temperatures. Due to unknown impact of their three dimensional structure on miscibility and stabilization of drug against crystallization, each of these polymers possesses great potential for use in various drug delivery applications. / Master of Science

No / A series of lipoamino acid-based glycolipids were synthesised. Suitably derivatised lipoamino acid derivatives were prepared and conjugated to monosaccharides (including glycosyl azides, isothiocyanates, thiols and sulphones) to yield novel O-, N-, S- and C-linked glycolipids in good yields. Their potential to improve the oral absorption of piperacillin is reported.

This project introduces a novel ultrasonic shearing-based fabrication method for synthesizing nanobubbles, which can then be utilized as a platform for any theranostic applications in clinical medicine, such as drug/gene delivery systems. Our standard in situ sonochemical synthesis of nanobubbles incorporates a perfluorocarbon gas core (300 μl) and an albumin outer shell, which are then incorporated into phosphate-buffered saline (4 ml) and later sonicated with a US probe. The initial optimization phase consisted of experimenting with various amounts of human serum albumin (HSA), which stabilizes the nanobubble gas core. Of the parameters (20 mg, 40 mg, and 80 mg HSA), 40 mg HSA significantly outperformed (pin vivo imaging nanobubbles in the liver. A continuation of this project will continue to optimize and expand on the theranostic applications of the US sheared nanobubbles in vivo and ex vivo in osteoporosis and the bone.

Eye disorders are on the rise as a result of an ageing population, an increasing obesity problem and a growth in the number of diabetic patients. Conventional ophthalmic formulations do not maintain therapeutic drug concentration in the target tissues for a long duration due to the physiological and anatomical eye barriers. Novel delivery systems such as nanoparticles have been explored to enhance the delivery of therapeutic agents to the eye. These delivery systems have in general been assessed using in-vivo animal models, despite ethical concerns for animal wellbeing. The aims of this thesis were to synthesise and characterise four amphiphilic polymers, subsequently prepare and characterise four nano sized polymeric self-assemblies loaded with triamcinolone acetonide (TA), develop an in-vitro porcine eye model and to evaluate the permeation of nano sized self-assemblies using the developed model. Four comb-shaped amphiphilic polymers (Pa5, Pa5-MPEG, Ch5 and Da10) were synthesised with a high yield (>81%) and good reproducibility. These polymers formed spontaneous positive self-assemblies in aqueous media (114-314 nm). The mean hydrodynamic diameters of the positive spontaneous self-assemblies entrapping TA were in the range of 200–334 nm loading high concentrations (455-1263 μg mL-1) of TA, much greater than the TA inherent aqueous solubility or concentrations achieved using conventional solubilisers. A porcine in-vitro eye model was developed to assess drug permeation through anterior and posterior ocular tissues. The model was partially validated using tritiated water and a series of hydrophilic markers with increasing molecular weights. The integrity of porcine ocular tissue was checked by monitoring the permeation of tritiated water to ensure the membrane intactness. Tritiated water permeation at 15 min was exploited as a potential method to normalise drug flux, as tritiated water percentage permeation at 15 min had an inverse relationship with tissue thickness (R2 = 0.66), to reduce the inherent variability between tissue samples thus increasing the accuracy of the in-vitro eye model. Four markers (fluorescein sodium salt, 4, 10 and 20 kDa FITC-dextran) were used for the purpose of investigating the effect of increasing molecular weight on ocular tissue permeability. The permeability of the markers displayed an inverse relationship and abrupt decline with Mw in terms of the permeability through scleral and corneal tissues of human and porcine and the molecular weight of the markers. The developed porcine in-vitro eye model showed good correlation with the human in-vitro model providing strong evidence it can be used to screen potential formulations before testing in-vivo. The TA loaded self-assemblies and a few chemical enhancers (glutamic acid, tween 80, chitosan, Pa5 and elevate temperature (45 °C)), selected to assist drug delivery via two routes (paracellular and transcellular), were tested using the developed in-vitro eye model. The results showed there was no marker permeation enhancement effect in porcine and human ocular tissues using chemical enhancers. In summary, a porcine in-vitro eye model was developed to assess hydrophobic and hydrophilic penetrant permeation across anterior and posterior ocular tissues. The porcine in-vitro eye model showed good correlation with the human in-vitro model providing strong evidence that the porcine in-vitro eye model can be used to screen potential formulations before testing in-vivo using the porcine model which ultimately might correlate well with the in-vivo human responses. Although TA self-assemblies did not significantly increase drug flux through human or porcine scleral tissues, it might be of interest for ophthalmic topically administered formulations due to their positive charge and small nano size.

Student Number ; 9901384G - MPharm dissertation - School of Pharmacy and Pharmacology - Faculty of Health Sciences / A lyophilized polymeric wafer system was formulated for the provision of rapid drug release in the oramucosal region. Lyophilization produced a porous sponge-like matrix which allowed simulated saliva to be rapidly imbibed into the hydrophilic structure. This surge of simulated saliva resulted in rapid disintegration of the wafer. Hydroxypropyl cellulose (HPC) was selected as the polymeric platform based on its low gelation potential. Other excipients incorporated into the system were lactose and mannitol as diluents, and glycine as a collapse protectant. A Face Centred Central Composite Design was chosen to establish the significant effects of the independent formulation variables on the physicochemical and physicomechanical properties of the wafer. The formulation variables investigated were, HPC concentration, type of diluent (lactose, mannitol or mixture), concentration of diluent, quantity of glycine and fill volume. An analysis of these variables elucidated the influential factors that may be controlled to form an ‘ideal’ wafer. The concentration of HPC significantly affected the disintegration rate (p=0.003), influx of simulated saliva (p=0.011) and friability (p=0.023). The quantity of diluent present in the system also had significant effect on matrix tolerance (p=0.029) and friability (p=0.032). Statistical optimization was undertaken using stepwise forward and backward regression, and Artificial Neural Networks to predict the ideal combination of the independent variables that would produce an ideal formulation. This wafer was required to produce a matrix disintegration of 3.33%/s, friability of 0.1% loss and maximum matrix resilience. Formulations manufactured with and without model drug, diphenhydramine hydrochloride, reflected no significant differences in their physicomechanical and physicochemical properties. In an attempt to expand the scope of this technology, a preliminary investigation was undertaken to develop a prolonged release wafer system. This was successfully achieved trough the application of crosslinking technology. It was possible to achieve drug released over a period of 6 hours.

Ng, Yiu Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 99-114). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Content --- p.6 / List of abbreviations and symbols --- p.10 / Chapter Chapter 1 - --- Introduction --- p.13 / Chapter 1.1 --- Introduction to nanoparticles (NPs) --- p.13 / Chapter 1.2 --- How to treat solid cancers using nanoparticle drugs --- p.17 / Chapter 1.3 --- What is Chitosan (CS)? --- p.22 / Chapter 1.4 --- Possible peptide candidates to be trapped --- p.26 / Chapter 1.4.1 --- Luffin PI - Ribosome inactivating peptide --- p.26 / Chapter 1.4.2 --- Buforin lib (Bllb) - Antimicrobial peptide --- p.27 / Chapter 1.5 --- Aims of study --- p.30 / Chapter Chapter 2 - --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.2 --- Methods --- p.31 / Chapter 2.2.1 --- Construction and expression of Luffin P1 --- p.31 / Chapter 2.2.2 --- Circular dichroism spectroscopy --- p.32 / Chapter 2.2.3 --- Static light scattering --- p.33 / Chapter 2.2.4 --- In vitro N-glycosidase assay --- p.34 / Chapter 2.2.5 --- Preparation of CS particles --- p.34 / Chapter 2.2.5.1 --- Preparation of positive CS NPs --- p.34 / Chapter 2.2.5.2 --- Preparation of negative CS NPs --- p.35 / Chapter 2.2.5.3 --- Preparation of buforin lib incorporated NPs --- p.35 / Chapter 2.2.5.4 --- Preparation of Cy5 incorporated NPs --- p.36 / Chapter 2.2.6 --- Characterization of CS NPs --- p.36 / Chapter 2.2.7 --- Buforin lib (Bllb) encapsulation efficiency and loading capacity --- p.36 / Chapter 2.2.8 --- In vitro release study --- p.37 / Chapter 2.2.9 --- Confocal Microscopy --- p.37 / Chapter 2.2.10 --- Cytotoxicity assay --- p.38 / Chapter 2.2.11 --- Statistical analysis --- p.38 / Chapter Chapter 3 - --- "Cloning, expression, purification and structural characterization of Luffin PI" --- p.39 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Results --- p.41 / Chapter 3.2.1 --- Construction of Luffin PI plasmid --- p.41 / Chapter 3.2.2 --- Expression and purification of Luffin PI --- p.41 / Chapter 3.3.3 --- Molecular weight and secondary structure determination of Luffin PI --- p.43 / Chapter 3.3.4 --- 3D solution structure of Luffin PI --- p.45 / Chapter 3.3.5 --- In vitro N-glycosidase activity of Luffin PI --- p.49 / Chapter 3.3 --- Discussion --- p.51 / Chapter Chapter 4 - --- Generation of positively charged CS particles and Bllb incorporation --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Results --- p.62 / Chapter 4.2.1 --- Positively charged CS NPs generation --- p.62 / Chapter 4.2.2 --- Bllb incorporated +ve CS NPs generation --- p.68 / Chapter 4.2.3 --- In vitro release study --- p.70 / Chapter 4.2.4 --- In vitro cytotoxicity test --- p.72 / Chapter 4.3 --- Discussion --- p.74 / Chapter Chapter 5 - --- Generation of negatively charged CS particles and Bllb incorporation --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Results --- p.85 / Chapter 5.2.1 --- -ve CS NPs generation --- p.85 / Chapter 5.2.2 --- -ve CS-Bllb NPs generation --- p.88 / Chapter 5.2.3 --- In vitro release study --- p.91 / Chapter 5.2.4 --- Localization study of -ve CS-Bllb NPs --- p.93 / Chapter 5.2.5 --- In vitro cytotoxicity test --- p.96 / Chapter 5.3 --- Discussion --- p.98 / Chapter Chapter 6 - --- Conclusion and future work --- p.108 / Copyright --- p.110 / References --- p.111

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