The objective of this project was to develop new controlled drug delivery systems using nanomeric particles and characterize the delivery of drugs into cells in real time by digital fluorescence imaging microscopy techniques. The project is based on the idea that it could be possible to improve efficacy of drug molecules when encapsulated in nanometer-sized particles. Due to their small dimensions the particles could permeate through cells and tissues and even through the blood brain barrier. The anti-cancer drug Doxorubicin was encapsulated into biodegradable Poly (DL-lactideco- glycolide) (PLGA) nanoparticles by simple nanoprecipitation method. The small size of these particles (<200nm) could be beneficial to realize passive tumor-targeted drug delivery through enhanced permeability and retention (EPR) effects. These drug-containing particles showed a sustained release profile. Fluorescence images indicated that these particles can be internalized by human breast cancer MCF-7 cells by non-specific endocytosis. The bioactivity of the drugs was also tested against cell culture. The results indicated that DXR-loaded PLGA nanoaprticles could be used to deliver Doxorubicin into breast cancer cells.

When developing new techniques to analyze neuro-chemical microenvironments, it is important to realize the incredible variability in the cellular content and the response to stimulation between cells and within a single cell. Conventional analysis techniques yield an average result to describe the content and function of cells. This approach often misses important information since the onset of pathological conditions is always initiated in a small number of cells. New minimally invasive single cell analysis techniques are required for single cell studies in order to gain new insights and understanding of cells' functions. The objective of my Ph.D. study was to fabricate, characterize, and apply submicrometric fluorescence sensors for the analysis of neuron cells. This dissertation will report the fabrication of miniaturized fluorescence sensors for Ca2+, pH and Zn2+ analysis. In the first approach, liposomes (phospholipid vesicles) were used as miniaturized containers for fluorescent sensing reagents. Liposome-based fluorescence sensing technology offers several advantages over commonly used fluorescence sensing techniques including high spatial resolution, protection of the sensing dye from quenchers and high biocompatibility. However, liposome based sensors were found to be unstable in the cellular environment. The second approach was to synthesize submicrometric particle-based fluorescence sensors named lipobeads to replace the fluorescent liposomes in cellular studies. Lipobeads are polystyrene particles that are coated with a phospholipid membrane. One unique advantage of fluorescent sensing lipobeads is the ability to immobilize hydrophobic indicator molecules in the phospholipid membrane. This enables the use of these indicators in aqueous media since the lipobeads are fully water miscible. The lipobeads also proved to be highly biocompatible in cellular studies. This is attributed to their phospholipid bilayer membrane, which is similar in structure to cell membranes. The dissertation will describe the analytical properties of fluorescence sensing lipobeads and their application in studying zinc ion release and pH changes near neuron cells under physiological conditions, conditions of neuronal injury and stress and acidic cortical spreading depression during stroke like conditions.

Le statut ganglionnaire a la valeur pronostique la plus importante chez les patientes atteintes d’un cancer du sein, le taux de survie étant corrélé au nombre de ganglions envahis. Dans le traitement des cancers du sein opérables d'une taille inférieure à 3 cm, la technique du ganglion sentinelle (GS) remplace le curage ganglionnaire de stadification. Toutefois, cette technique nécessite l’emploi simultané de radioisotopes et de colorants physiologiques présentant divers effets secondaires. Ainsi, notre stratégie a été de tester, sur modèle pré-clinique de nouveaux traceurs fluorescents, les Quantum Dots (QDs), pour la technique du GS. Une étude sur la détection in vivo du ganglion axillaire (GA) par spectrofluorimétrie fibrée après injection sous-cutanée (s.c.) de QDs émettant dans le rouge a été réalisée et leur quantification ex vivo a été effectuée par spectrofluorimétrie jusqu’à 24 h. Ce travail a montré l’accumulation rapide et sélective des QDs dans le GA chez la souris. Une étude sur la localisation in vivo de 2 ganglions régionaux par imagerie de fluorescence a été réalisée suite à l’administration s.c. de QDs émettant dans le proche infrarouge (PIR) ainsi qu’une étude de biodistribution ex vivo par spectrométrie de masse (ICP-MS). Les résultats obtenus ont montré que l’imagerie de fluorescence pouvait être utilisée après injection de QDs pour localiser et suivre leur accumulation dans les ganglions superficiels chez la souris saine. Pour se rapprocher de la clinique, le repérage in vivo du GS axillaire a été investi sur modèle tumoral murin de cancer du sein par imagerie de fluorescence après administration s.c. de QDs émettant dans le PIR. Deux techniques de détection des métastases ganglionnaires ont été utilisées puis comparées : l’histologie conventionnelle et la RT-PCR. Tous les GS axillaires ont été détectés in vivo par imagerie, sauf un GS qui était complètement envahi par les cellules tumorales et la meilleure incidence de métastases ganglionnaire a été observée avec la RT-PCR. Ainsi, les QDs pourraient être utilisés comme substitut des marqueurs actuellement employés dans la technique du GS pour les cancers du sein. / Lymph node (LN) status is the most important prognostic factor in breast cancer patients and a determinant predictor of recurrence and survival. In the treatment of operable breast cancers with a size inferior of 3 cm, the sentinel lymph node biopsy (SLNB) substitutes the axillary LN dissection. However, this technique requires the simultaneous use of a radiocolloid and a physiologic dye, which present several secondary effects. Thus, our strategy was to test in preclinical model the new fluorescent markers, Quantum Dots (QDs) for SLNB. A study on in vivo axillary LN (ALN) mapping by light induced fluorescence was performed after subcutaneous (s.c.) injection of red-emitting QDs and their ex vivo quantification was realized by spectrofluorimetry for up to 24 h. We showed a fast and selective accumulation of QDs in ALN in healthy mice. An in vivo study on localization of 2 regional LNs by fluorescence imaging was carried out after the s.c. administration of near-infrared (NIR) emitting QDs along with ex vivo biodistribution study by mass spectroscopy (ICP-MS). Obtained results shown that fluorescence imaging can be used after QDs injection to map and follow their accumulation in superficial LNs in healthy mice. To approach the human clinic in a best possible way, sentinel ALN mapping was investigated after s.c. delivery of NIR emitting QDs in murine breast cancer model by fluorescence imaging. Two techniques for LN metastasis detection were used and compared: conventional histology and RT-PCR. All sentinel ALNs were located in vivo by imaging except one ALNs which was completely invaded by cancer cells and best incidence of LN metastasis was registered by RT-PCR (60 %). Thus, QDs could be used as a substitute for the markers currently employed in SLNB in breast cancers.

David Lai. / Thesis -- Chinese University of Hong Kong. / Bibliography: l. 103-104. / Acknowledgement --- p.v / Abstract --- p.vi / Format of the Thesis --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Experimental Techniques / Chapter 2.1 --- Introduction --- p.10 / Chapter 2.2 --- Arrangement for Fluorescence Spectrum Measurements --- p.10 / Chapter 2.3 --- Fluorescence Spectrum Measurements by Photographic Method --- p.11 / Chapter 2.4 --- Arrangement for Fluorescence Decay Rate Measurements --- p.11 / Chapter Chapter 3 --- Soda-lime Glass Matrix / Chapter 3.1 --- Introduction --- p.17 / Chapter 3.2 --- Theory --- p.19 / Chapter 3.3 --- Samples --- p.23 / Chapter 3.4 --- Experiments and Results --- p.26 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Fluorescence Spectra --- p.35 / Chapter 3.5.2 --- Decay Rate Measurements --- p.39 / Table (3-1) --- p.43 / Table (3-3) --- p.43 / Table (3-2) --- p.44 / Chapter Chapter 4 --- Lithium Phosphate Glass / Chapter 4.1 --- Introduction --- p.45 / Chapter 4.2 --- Samples --- p.46 / Chapter 4.3 --- Experiments and Results --- p.47 / Chapter 4.4 --- Discussion --- p.54 / Table (4-1) --- p.64 / Table (4-2) --- p.65 / Table (4-3) --- p.66 / Table (4-4) --- p.66 / Chapter Chapter 5 --- Hydrogen Oxide (H20) and Deuterium Oxide (D20) / Chapter 5.1 --- Introduction --- p.67 / Chapter 5.2 --- Theory --- p.69 / Chapter 5.3 --- Experiments and Results --- p.71 / Chapter 5.4 --- Discussion --- p.71 / Table (5-1) --- p.78 / Table (5-2) --- p.79 / Chapter Chapter 6 --- Conclusion --- p.80 / Appendix I --- p.82 / Appendix II --- p.83 / Appendix III --- p.84 / Appendix IV --- p.85 / Appendix V --- p.86 / Appendix VI --- p.90 / Appendix VII --- p.91 / Appendix VIII --- p.95 / Appendix IX --- p.96 / Appendix X --- p.102 / References --- p.103

In recent years, fluorescence imaging has become an indispensable tool for the exploration of biological processes, demonstrating both molecular specificity and high spatial and temporal resolution. Despite the significant progress made in this field, a number of challenges still exist which, if addressed could potentially result in the transformative development of fluorescent imaging for a plethora of biological applications. This may include the development of new fluorescent probes for the detection of unknown analytes, or the improvement of existing probes in order to enhance their properties. In this research, a fluorescent probe for the detection of hydrogen sulphide was repurposed for use as a ‘first of its kind’ fluorescent probe for the detection of hydroxylamine. In addition, the known peroxynitrite-mediated oxidation of boronic acid to phenol has been exploited for the development of a range of reaction based fluorescent probes. Initially non-fluorescent, each probe is ‘turned on’ in the presence of peroxynitrite, resulting in the formation of a highly fluorescent phenol derivatives. Such probes have been successfully evaluated during cell imaging experiments; demonstrating clear potential in the field of medical diagnostics. Specific applications may include ‘oxidative stress’, neurodegenerative disease and the evaluation of drug efficacy in the treatment of Alzheimer’s disease.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 134-151). Also available in electronic version.

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Herein is presented the collective experimental record of research performed in the Laboratory for Biological Surface Science. These investigations are generally classified under the category of bioanalytical surface science and include the following projects. Chapters III and IV describe the creation of a microfluidic device capable of generating fixed arrays of concentration gradients. Experimental results were matched with computational fluid dynamics simulations to predict analyte distributions in these systems. Chapters V and VI demonstrate the discovery and utility of photobleaching fluorophores for micropatterning applications. Bleached fluorophores were found to rapidly attach to electron rich surfaces and this property was used to pattern enzymes inside microfluidic channels in situ. Finally, Chapter VII exhibits a method by which solid supported lipid bilayers can be dried and preserved by specifically bound proteins. The intrinsic property of lateral lipid mobility was maintained during this process and a mechanism by which the protein protects the bilayer was suggested.

Secretory proteins that are unable to assemble into native proteins in the endoplasmic reticulum (ER) are transported back into the cytosol for degradation. Many cytosolic and ER resident proteins have been identified so far as being involved in this retrotranslocation process, but it is difficult to determine whether these proteins have a direct or indirect effect. Interpretations are further complicated if the loss of a specific protein is obscured by the presence of another protein that is partially or wholly redundant. To overcome these limitations, a mammalian in vitro system was developed that allowed to monitor retrotranslocation synchronously and in real time in the absence of concurrent translocation. To examine the roles of different components in ER-associated degradation (ERAD), well-defined and homogeneous mammalian ER microsomes were prepared biochemically by encapsulating a fluorescent-labeled ERAD substrate with specific lumenal components. After mixing ATP, specific cytosolic proteins, and specific fluorescence quenching agents with microsomes, substrate retrotranslocation was initiated. The rate of substrate efflux from microsomes was monitored spectroscopically and continuously in real time by the reduction in fluorescence intensity as the fluorescent substrates passed through the ER membrane and were exposed to the quenching agents. Retrotranslocation kinetics were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase, or all cytosolic proteins with only the 19S proteasome cap. Retrotranslocation was blocked by affinity-purified antibodies against Derlin1, but not by affinity-purified antibodies against Sec61α or by membrane-bound ribosomes. Since the substrate also photocrosslinked Derlin1, but not Sec61α or TRAM, retrotranslocation of this ERAD substrate apparently involves Derlin1, but not the translocon. By labeling either the C- or N-terminus, it was revealed that the N-terminus of one ERAD substrate leaves the ER lumen first. This finding suggests that the protein is retrotranslocated as a linear polymer in a preferred direction. When RRMs were reconstituted with a fluorescent-labeled ERAD substrate and various ions. Ca2+ ions in the ER lumen increased the rate and extent of retrotranslocation, while Ca2+ ions in the cytosol decreased retrotranslocation. This approach therefore provides the first direct evidence of the involvement and importance of specific ionic requirements for ERAD.

Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phosphoenolpyruvate (PEP), which act to activate or inhibit, respectively, by changing the substrate (Fru-6-P) affinity of the enzyme. Both ligands bind to the same allosteric site in EcPFK. Therefore, the questions we want to address are how these two molecules regulate EcPFK and how the allosteric signal is propagated throughout the enzyme. EcPFK has 28 potential site-site interactions. These interactions in turn derive from multiple copies of 6 potentially unique homotropic interactions and 4 potentially unique heterotropic interactions. Making hybrid tetramer of EcPFK is used to isolate a single heterotropic interaction. To improve the yield of the 1:3 hybrid, the in vivo hybrid formation method was developed. Four heterotropic interactions were isolated by this manner and re-evaluated. The same kinetics characteristics were obtained for each 1:3 hybrid from both the in vivo and in vitro method. To address the question of how the allosteric signal is transmitted throughout EcPFK, we identified residues (G184, Asp59 and S157) that are important for the allosteric regulation for both PEP inhibition and MgADP activation. The impact of each mutation on individual interaction is unique and also suggests that the structural basis for PEP inhibition is different from that for MgADP activation. Most importantly, since the sum of each heterotropic interaction with a modification in only one subunit is equal to the total heterotropic interaction with a modification in all four subunits, this result indicates that the heterotropic allosteric signal transmission is realized in a single subunit. The 23Ã heterotropic interaction, which contributes the most to the PEP inhibition, was chosen to study the dynamic properties. Fluorescence was used to study the dynamic perturbations of the 23Ã interaction upon ligand binding. Taking advantage of the hybrid formation strategy and the tryptophan-shift mutagenesis method, a tryptophan residue can be placed at different individual locations throughout the native subunit containing the 23Ã heterotropic interaction. The steady-state anisotropy and lifetime measurement at each tryptophan position indicate that the 23Ã allosteric interaction involves the perturbation of side-chain dynamics both near and quite far away from the respective ligand binding sites.