Glioblastoma is the most common adult malignant primary brain tumor with one of the worst prognosis. With a survival of 10 to 12 months, glioblastoma remains one of the most challenging disease to treat. The standard treatment method involves maximal possible resection of the tumor followed by radiation and chemotherapy. However, the short half-life of most chemotherapeutic drugs, high systemic toxicity and inability to cross the blood brain barrier inhibits effective delivery of the chemotherapeutics to the tumor. An ideal drug delivery system can reach the tumor site with high efficiency and continuously release the drug at the tumor site for an extended period. Adult stem cells including neural stem cells (NSC) and mesenchymal stem cells (MSC) have inherent tumor trophic properties allowing for site-specific delivery of chemotherapeutics. They can also be genetically engineered to secrete the chemotherapeutic drug continuously making them ideal candidates for cell-based delivery system for treating glioblastoma. MSC have been isolated from a wide range of sources including bone marrow, umbilical cord, adipose tissue, liver, multiple dental tissues and induced pluripotent stem cells. MSC are also easily amenable to viral modification allowing for easy manipulation to produce chemotherapeutic drugs. Additionally, more than 350 clinical trials using MSC have successfully established the safety of using MSC for cell-based therapies. Collectively these factors have led to the widespread use of MSC in cancer therapy. MSC have been successfully transduced to produce chemotherapeutic drugs to treat glioma, melanoma, lung cancer, ovarian cancer and breast cancer. Despite the multitudes of advantages that cell therapy provides they are limited in three main domains (1) Low cell retention and survival at the site of the tumor (2) In ability to co-deliver multiple therapeutics and (3) In ability to deliver drugs other than peptide based drugs. This thesis details the work to engineer mesenchymal stem cells to tackle these three issues and develop a system that can increase the efficacy of glioblastoma treatment. To increase the cellular retention and survival we engineered MSC to form multicellular spheroids and cell sheets. To co-delivery multiple therapeutics we engineered MSC to form MSC/DNA-templated nanoparticle hybrid cluster to co-deliver drugs for cancer therapy. The system showed superior performance due to the increased retention of the cells and nanoparticle at the tumor site. Finally, to deliver drugs other peptide based we engineered graphene oxide cellular patches for mesenchymal stem cells. Graphene oxide can carry diverse therapeutics and can kill the cancer cells without affecting the cellular viability of MSC.

No description available.

The potential of magnetic nanoparticle-vesicle assemblies (MNP-V) as remote controlled drug delivery platforms capable of inducing cellular responses under magnetic stimuli has been previously demonstrated in the Webb group at the University of Manchester. To create these magnetoresponsive nanomaterials biotin-avidin and Cu-histidinyl multivalent recognition were employed. This thesis describes an exploration of the potential of thiol-thioester exchange reactions (leading to native chemical ligation, NCL) to create magnetoresponsive materials, which potentially have applications in biomedicine. Firstly, iron oxide magnetic nanoparticles have been synthesised using a thermal co-precipitation method followed by chemical modification with sulfhydryl motifs for use as smart biomaterials. Knowing that the behaviour and reactivity of nanoparticles is highly influenced by their physicochemical properties, a thourough characterisation of these particles has been obtained. Secondly, during this project, several thioester derivatives have been synthesised that can be incorporated into the membranes of 800 nm liposomes. Among these, the spectrophotometric properties of synthetic lipid 38 allowed the investigation of trans-thioesterification rates with cysteinyl functionalities, both in solution and at the phospholipid membrane interface of liposomes. Product identification has been achieved using mass spectrometry and 1H-NMR spectroscopy. Finally, the conditions required to induce the release of a dye (e.g. 5(6)-CF) from MNP-V upon exposure to an AMF pulse have been established. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases has been investigated as interesting payload due to its fluorescent and anti-viral properties.

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

The first polysulfenamides were synthesized with S-N and N-S-N bonds along the backbone. We demonstrated that sulfenamides were stable in polar protic and aprotic solvents, but degraded rapidly when exposed to acidic conditions. Microparticles were fabricated from polysulfenamides with S-N bonds, their surfaces were readily functionalized, and they were internalized by cells allowing for intracellular delivery of their cargo. These microparticles were also stable at physiological pH, degraded under acidic conditions, and possessed minimal toxicity towards cells. This work demonstrated that polysulfenamides form the basis for a new set of polymers for drug delivery that greatly differ from prior work in this field. New biodegradable polymers with N-S-N bonds along the backbone were synthesized. These were the first polymers with these bonds and possessed many of the same characteristics as polymers synthesized with S-N bonds. The synthesis and characterization of comb block copolymers with arms composed of poly(lactic acid), poly(butyl acrylate), and poly(styrene-b-vinylpyridine) were described. The self-assembled morphologies in the solid state of comb tri- and tetrablock copolymers with poly(styrene) were also described. These assemblies demonstrated that well-ordered and complex morphologies were assembled from these polymers. The steric effect of substitutions on oxanorbornenes in ring opening metathesis polymerization (ROMP) was investigated. Oxanorbornenes substituted with methyls at the bridgehead positions showed limited reactivity with the Grubbs first and second generation catalysts and the Grubbs first generation methylidene catalyst.

A methodology was developed to conduct diagnostic performance assessment and simulation of alternative delivery scenarios in pressurized irrigation distribution networks. It consists of three components, an agro-hydrologic model able to forecast peak water demand hydrographs, a hydraulic model with capability of simulating the network behavior under different flow configurations, and a set of performance indicators for conducting assessments of performance achievements relative to specified targets. As a preliminary work, the current delivery schedule of an existing pressurized irrigation network (system 1) and the resulting effects on crop irrigation management were analyzed by simulating soil water balance and irrigation scheduling at field level. Simulations allowed analyzing the on-farm irrigation management under the current rotation deliveries, and comparing it with an alternative flexible irrigation scheduling to maximize crop yields. Results at field level were up-scaled to the entire command area of the system, showing the usefulness of soil water balance and of irrigation scheduling as analytical tools to demonstrate the inconsistency between the current water delivery and crops’ and farmers’ requirements. This preliminary work also allowed highlighting the need for modernizing the irrigation management in the first of the two study areas considered for the present research work (system 1 located in the province of Taranto, southern Italy). An existing agro-hydrological model conceived to forecast water demand hydrographs in pressurized delivery networks was enhanced through several refinements and amendments of the computation algorithms. The refined model was applied for validation at different management levels on an existing pressurized irrigation system (system 2) located in the province of Foggia, southern Italy, where water withdrawals by farmers and the main hydraulic parameters are recorded on a continuous basis for monitoring purposes. Results from validation showed that the model is capable of forecasting with good accuracy the timing of peak-demand periods, the seasonal demand irrigation volumes, as well as the hydrographs of hourly flow rates demanded by farmers during these peak periods, especially when it is applied to large multi-cropped command areas. Performance indicators, originally conceived for diagnostic assessment in canal systems, were modified for application to pressurized distribution networks, and reference standard values were proposed. These indicators were then applied for validation to the second study area (irrigation system 2), where records of water deliveries are available, and showed their usefulness for diagnostic performance assessments.

Abstract not available

<p>Pharmacotherapy for Parkinson’s disease (PD) is based on levodopa, the most effective dopaminergic drug. The development of motor complications constitutes the major challenge for new or refined therapies.</p><p>To evaluate the impact of levodopa pharmacokinetics on motor function, an observational study in the patients’ home environment was carried out. A high variability in plasma levodopa levels was found in all patients, irrespective of treatment regimen. The impact of levodopa pharmacokinetics was further studied in a crossover trial comparing sustained-release tablets and continuous daytime intestinal infusion. Infusion produced significantly decreased variability in plasma levels of levodopa, resulting in significantly normalised motor function. A permanent system for long-term levodopa infusion has been developed and 28 patients have been followed for 87 patient-years. Motor response was generally preserved during the long-term observation period, implying that there is no development of tolerance to infusion therapy. Levodopa tablets are normally used in multiples of 50 or 100 mg, thus a rough estimate of individual dosage. A new concept for individualising levodopa/carbidopa doses with microtablets of 5/1.25 mg is under development. An electronic drug-dispensing device for administering the microtablets was tested on patients with PD. All were able to handle the dispenser and most were interested in future use of the concept. Self-assessment of symptoms is accurate in PD, but traditional paper diaries are associated with low compliance. A wireless electronic diary was compared with a corresponding paper diary. The time-stamped and thus completely reliable patient compliance was 88% with the electronic diary.</p><p>To conclude, pharmacokinetics of levodopa is the major determinant for motor fluctuations in PD. Every effort to individualise dosage and to smooth out the fluctuations in levodopa concentrations should be made, e.g. by means of microtablets or enteral infusion. Electronic patient diaries for real-time data capture are suitable for PD studies.</p>

Graduation date: 2003

Gene therapy has emerged as a field to modulate cell functions by introducing genes of interest to target cells. An emerging focus in this field is to employ non-viral vectors to deliver immunosuppressive cytokines to dendritic cells (DCs) to attenuate damaging immune responses. DCs serve as potential targets for suppression of T cell responses. In this work, we investigated the ability of polycation-modified polymeric particles complexed with interleukin-10 (IL-10) gene to modulate DCs. The delivery systems (designated as PSO10H6 and PLGAO10H6) were formed by coating cationic peptide O10H6 (O: ornithine; H: histidine) on the polystyrene (PS) and poly (lactic-co-glycolic acid) (PLGA) particulates. A mouse IL-10 encoding plasmid (pIL-10) was loaded on the surface of PSO10H6 and PLGAO10H6 via ionic interactions. Physical characterization of these particles revealed stable colloidal dispersions (diameters: 297.2&#177;14nm in PLGAO10H6-pIL-10 and 126.0&#177;8nm in PSO10H6-pIL-10). DNA molecules carried by PSO10H6 and PLGAO10H6 were protected from serum digestion. Results from in vitro gene transfection studies showed two-fold enhancement of IL-10 expression in bone marrow-derived DCs transfected with PSO10H6-pIL-10 and PLGAO10H6-pIL-10 compared to untransfected DCs. Their suppressive functions were evaluated in an in vitro mixed lymphocyte model. Results indicated that PSO10H6-pIL-10 and PLGAO10H6-pIL-10 modified DCs elicited weakest proliferation of allogeneic bulk T cells as well as CD4 and CD8 T cells among all the delivery modes. Using cell-embedded Matrigel as a surrogate graft, we showed that IL-10 gene-modified DCs suppressed host cell infiltration in vivo. These data suggested PSO10H6-pIL-10 and PLGAO10H6-pIL-10 deliver an overriding suppressive signal to T cells. Further studies revealed T cells stimulated by the IL-10 gene-modified DCs exhibited characteristics of regulatory T (Treg) cells, as evident by up-regulation of a Treg cell marker forkhead-type transcription factor 3 (Foxp3). This result was concomitant with an increase in of transforming growth factor beta (TGF-beta) production. &lt;br&gt;Taken together, this work demonstrated that PSO10H6 and PLGAO10H6 are effective in delivering pIL-10 to modulate DCs to suppress T cell responses. Collectively, the results raise the prospects of using PSO10H6 and PLGAO10H6 as vectors to deliver immunosuppressive genes to modulate T cell responses in vivo. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmaceutics / PhD / Dissertation