Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references (p. 94-113).

abstract / Pharmacology and Pharmacy / Master / Master of Philosophy

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references.

Liposomes have important potential as drug delivery vehicles. However, in order to realize this potential, much basic research is required to elucidate the interactions experienced by liposomes in vivo. In this thesis two aspects of these interactions are investigated: the influence of vesicle size and lipid composition on the biodistribution observed in vivo; and the interaction of liposomes with plasma proteins. In order to determine the in vivo behavior of liposomal systems, a new vesicle marker is synthesized (¹²⁵I-tyraminyl-inulin, ¹²⁵ITI) and tested in vivo. It is shown that this probe satisfies the necessary criteria for an accurate marker of liposome behavior, and is superior to probes used by other workers in terms of accuracy, convenience, high specific activity, low tissue quenching and cost. The use of ¹²⁵ITI as a vesicle marker allows accurate measurements to be made with lower doses of liposomes than previously employed. The influence of vesicle size, composition, and dose on the blood residency times, leakage and tissue distributions of vesicles was therefore investigated at these lower doses, employing a cannulation procedure to monitor vesicles. It is demonstrated that the clearance of vesicles from the circulation exhibits biphasic kinetics. The relative number of vesicles cleared during the early phase (halflife <20 min) is decreased by increasing the vesicle dose or decreasing the size. The behavior of small vesicles produced by extrusion is also investigated, and the in vivo behavior of these systems is shown to be equivalent to conventional sonicated systems. The second part of this thesis investigates the binding of plasma proteins to vesicles in vitro. It is shown that vesicles bind a large number of plasma components and that the binding is strongly dependent on the surface charge of the vesicle. Some of the proteins have been tentatively identified with 2-D electrophoresis and several were positively identified via immuno- autoradiography. A hypothesis is advanced regarding the role of plasma proteins in the fate of liposomes in vivo. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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

Lee, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 89-91). / Abstracts in English and Chinese. / Content --- p.i / Acknowledgments --- p.iv / Abstract --- p.V / Abbreviations and Acronyms --- p.vii / Publications Originated from the Work of this Thesis --- p.ix / Chapter Chapter 1- --- Introduction / Chapter 1.1 --- Nanoparticle-based drug delivery --- p.1 / Chapter 1.2 --- Magnetic nanoparticle --- p.5 / Chapter 1.3 --- Iron oxide nanoparticle --- p.6 / Chapter 1.3.1 --- Coprecipitation --- p.7 / Chapter 1.3.2 --- Hydrothermal reaction --- p.7 / Chapter 1.3.3 --- Sol-gel reaction --- p.8 / Chapter 1.3.4 --- Solvothermal reaction --- p.8 / Chapter 1.3.5 --- Architecture of iron oxide nanoparticles as drug carriers --- p.9 / Chapter 1.4 --- Supramolecular chemistry involved in controlled release drug delivery system --- p.10 / Chapter 1.5 --- Nano valve --- p.15 / Chapter 1.6 --- Aim of project --- p.17 / Chapter Chapter 2- --- Stimuli-Responsive Drug Delivery Nanosystems based on Fe3O4@SiO2@crown ether Nanoparticles / Chapter 2.1 --- Background --- p.19 / Chapter 2.2 --- Synthesis of the dibenzo-crown ethers --- p.21 / Chapter 2.3 --- Synthetic method of functionalized nanoparticles --- p.22 / Chapter 2.4 --- Characterization of dibenzo-crown ethers --- p.25 / Chapter 2.4.1 --- Nuclear magnetic resonance (NMR) spectroscopy --- p.25 / Chapter 2.4.2 --- Mass spectrometry (MS) --- p.27 / Chapter 2.4.3 --- Infrared (IR) spectroscopy --- p.28 / Chapter 2.5 --- Characterization of nanoparticles --- p.29 / Chapter 2.5.1 --- Transmission electron microscopy (TEM) --- p.29 / Chapter 2.5.2 --- Energy-dispersive X-ray (EDX) spectroscopy --- p.34 / Chapter 2.5.3 --- IR spectroscopy --- p.39 / Chapter 2.5.4 --- Thermogravimetric analysis (TGA) --- p.41 / Chapter 2.5.5 --- Nitrogen absorption/desorption isotherms --- p.44 / Chapter 2.6 --- Biological study of functionalized nanoparticles --- p.45 / Chapter 2.6.1 --- Cytotoxicity study --- p.45 / Chapter 2.6.2 --- Cell adhesion study --- p.46 / Chapter 2.6.3 --- Cell proliferation study --- p.47 / Chapter 2.6.4 --- Cellular uptake of nanoparticles --- p.50 / Chapter 2.7 --- Drug loading under different stimuli --- p.54 / Chapter 2.8 --- Drug release profile of nanoparticles --- p.61 / Chapter 2.9 --- MRI study of nanoparticles --- p.67 / Chapter 2.10 --- Conclusion --- p.69 / Chapter Chapter 3- --- Experimental Procedures / Chapter 3.1 --- General Information --- p.72 / Chapter 3.2 --- General procedure of synthesis of polyethers 3a-b --- p.73 / Chapter 3.2.1 --- Synthesis of 3a --- p.74 / Chapter 3.2.2 --- Synthesis of 3b --- p.74 / Chapter 3.3 --- General procedure of synthesis of diesters 4a-b --- p.75 / Chapter 3.3.1 --- Synthesis of 4a --- p.75 / Chapter 3.3.2 --- Synthesis of 4b --- p.76 / Chapter 3.4 --- General procedure of synthesis of dibenzo crown ether esters 5a-c --- p.77 / Chapter 3.4.1 --- Synthesis of 5a --- p.77 / Chapter 3.4.2 --- Synthesis of 5b --- p.78 / Chapter 3.4.3 --- Synthesis of 5c --- p.78 / Chapter 3.5 --- General procedure of synthesis of dibenzo-crown ethers la-c --- p.79 / Chapter 3.5.1 --- Synthesis of la --- p.80 / Chapter 3.5.2 --- Synthesis of lb --- p.80 / Chapter 3.5.3 --- Synthesis of lc --- p.81 / Chapter 3.6 --- Preparation of superparamagnetic Fe3O4 nanoparticle with an average diameter 120 nm --- p.81 / Chapter 3.7 --- Preparation of core/shell Fe3O4@SiO2 nanoparticle --- p.82 / Chapter 3.8 --- Preparation of Fe3O4@SiO2@meso(CTAB)-Si02 nanoparticle --- p.82 / Chapter 3.9 --- Preparation of Fe3O4@SiO2@meso(CTAB)-SiO2-NH2 nanoparticle --- p.83 / Chapter 3.10 --- Preparation of Fe3O4@SiO2@meso-SiO2@crown ether(a-c) nanoparticles --- p.83 / Chapter 3.11 --- Protocol of biological study of functionalized nanoparticles --- p.84 / Chapter 3.11.1 --- MTT protocol --- p.84 / Chapter 3.11.2 --- Cytotoxicity study --- p.84 / Chapter 3.11.3 --- Cell adhesion study --- p.85 / Chapter 3.11.4 --- Cell proliferation study --- p.85 / Chapter 3.11.5 --- Cellular uptake of functionalized nanoparticles --- p.85 / Chapter 3.12 --- Drug loading of functionalized nanoparticles --- p.86 / Chapter 3.13 --- Drug release profile of functionalized nanoparticles --- p.87 / Chapter 3.14 --- MRI study of nanoparticles --- p.87 / References --- p.89 / Appendix / List of Spectra --- p.A-1

The solubilities of three poorly water soluble drugs, phenytoin, diazepam and benzocaine, were measured in various cosolvent-water mixtures. The data were generally described by the relationship: log (S(m)/S(w)) = Σf₁σ₁ where S(m) is the solubility of the drug in the cosolvent-water mixture, S(w) is the solubility of the drug in water, f₁ is the volume fraction of cosolventi and σ₁ is the slope of the log(S(m)/S(w)) vs. f₁ plot. In most cases, some positive or negative deviation from the log-linear solubility equation is observed. The deviation is similar for all three drugs in many of the cosolvent-water mixtures. This suggests that the deviation is primarily due to interactions between the solvent components. However, it could not be predicted from any of the physical properties of the solvent mixtures. Changes in the solute crystal structure could not be identified as a source of nonideality. The deviations from the log-linear solubility equation may involve such factors as changes in solvent structure, hydrophobic hydration, density changes and hydrogen bonding differences between solute and cosolvent. The slopes, σ₁, of the solubilization plots were related to various indexes of solvent polarity including dielectric constant, solubility parameter, partition coefficient, surface tension and interfacial tension. The best correlations were obtained with measures of solvent cohesive forces such as interfacial tension and solubility parameter. In general, the solubilities in mixtures of aprotic cosolvents and water are higher than predicted by any of the polarity indexes. The slopes are thus related to the hydrogen bonding ability of the cosolvent as expressed by the density of proton donor and acceptor groups of the neat cosolvent. The slopes of the solubilization plots can be predicted from linear relationships with polarity indexes of the cosolvent. Therefore it is possible to estimate the slope, σ, in any cosolvent-water mixture from the solubilities in two solvents for a given drug. Furthermore, the solubility in any cosolvent water mixture can be estimated by combining the log-linear solubility equation with the estimated slopes.

by Chu Chi Yuen, Andrew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / ABSTRACT --- p.i / Chapter 1 --- INTRODUCTION --- p.3 / Chapter 1.1 --- Using Low density lipoprotein (LDL) as a drug carrier --- p.4 / Chapter 1.1.1 --- The structure of Low density lipoprotein (LDL) --- p.4 / Chapter 1.1.2 --- The metabolic pathway of LDL in human bodies --- p.4 / Chapter 1.1.3 --- The rationale for using LDL as a drug carrier --- p.7 / Chapter 1.1.4 --- Reconstitution of LDL with cytotoxic drugs --- p.9 / Chapter 1.1.5 --- Up and down regulation of LDL receptors --- p.11 / Chapter 1.2 --- Doxorubicin (DOX) --- p.12 / Chapter 1.2.1 --- Characteristics of DOX --- p.12 / Chapter 1.2.2 --- Drug actions of DOX --- p.14 / Chapter 1.2.3 --- The adverse side effects of DOX --- p.15 / Chapter 1.3 --- Multidrug resistance phenomenon in tumor cells --- p.17 / Chapter 1.3.1 --- The possible mechanisms of multidrug resistance --- p.19 / Chapter 1.3.2 --- The structure of P-glycoprotein --- p.20 / Chapter 1.3.3 --- The mechanisms of the P-glycoprotein --- p.22 / Chapter 1.3.4 --- Our aim in dealing with multidrug resistance --- p.22 / Chapter 2 --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Animals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Culture media --- p.25 / Chapter 2.1.4 --- Chemicals --- p.26 / Chapter 2.1.5 --- Culture of cells --- p.27 / Chapter 2.2 --- Methods --- p.29 / Chapter 2.2.1 --- In vitro studies --- p.29 / Chapter 2.2.2 --- In vivo studies --- p.44 / Chapter 3 --- RESULTS --- p.51 / Chapter 3.1 --- In vitro studies --- p.51 / Chapter 3.1.1 --- Preparation of LDL-DOX --- p.51 / Chapter 3.1.2 --- Comparison of the cytotoxicity of DOX and LDL-DOX on HepG2 cells --- p.59 / Chapter 3.1.3 --- Modulation of LDL receptors on HepG2 cells and ECV304 cells… --- p.63 / Chapter 3.1.4 --- The effect of combined treatment of LDL-DOX and hyperthermia on HepG2 cells --- p.84 / Chapter 3.1.5 --- The effect of LDL-DOX on resistant cell line R-HepG2 cells --- p.90 / Chapter 3.2 --- In vivo studies --- p.105 / Chapter 3.2.1 --- The comparison of organ distribution of LDL-DOX and DOXin BALB-c mice after administration --- p.105 / Chapter 3.2.2 --- The comparison of organ distribution of LDL-DOX and DOX in nude mice bearing HepG2 cells after adminstration --- p.108 / Chapter 3.2.3 --- Histological studies of heart of nude mice bearing HepG2 cells treated with DOX and LDL-DOX --- p.111 / Chapter 3.2.4 --- Myocardial injury measured by Lactate dehydrogenase (LDH) activity in nude mice bearing HepG2 treated with DOX and LDL- DOX --- p.117 / Chapter 3.2.5 --- The comparison of DOX and LDL-DOX on reducing the tumor sizes and weight in nude mice bearing HepG2 cells --- p.119 / Chapter 4 --- DISCUSSION --- p.122 / Chapter 4.1 --- In vitro studies --- p.122 / Chapter 4.1.1 --- Preparation of LDL-DOX complex --- p.122 / Chapter 4.1.2 --- The cytotoxicity ofLDL-DOX --- p.125 / Chapter 4.1.3 --- The combined treatment of hyperthermia and LDL-DOX --- p.129 / Chapter 4.1.4 --- The ability of LDL-DOX to circumvent muiltidrug resistance --- p.131 / Chapter 4.2 --- In vivo studies --- p.134 / Chapter 5 --- CONCLUSION --- p.136 / Chapter 5.1 --- Conclusion --- p.136 / Chapter 5.2 --- Future pospective --- p.139 / BIBLIOGRAPHY --- p.140

Aim of thesis: Immuno-stimulating complexes (ISCOMs) are particulate vaccine delivery systems composed of Quillaja saponins, cholesterol and phospholipid. ISCOMs are typically spherical cage-like structures with a diameter of 40 nm and carry a negative charge. Incorporation of the respective vaccine antigen into the particles generates more potent vaccines than a simple mixture of both vaccine components. This requires the antigen to display either hydrophobic domains or positive charges, which allow interaction with the ISCOM particles. However, not all antigens fulfil this requirement and modification of these becomes necessary. Hence, the aim of this study was to design novel preparations of ISCOMs with a positive charge, suitable for adsorption of native hydrophilic antigens and poly-nucleotides, and test their potential as a novel vaccine carrier platform. Methods: Two cationic lipids, DC-cholesterol and DOTAP, were selected to prepare the cationic modifications of ISCOMs. DC-cholesterol substituted for cholesterol in classical ISCOMs, whereas DOTAP substituted for their phospholipid component. The phase behaviour of colloidal systems containing Quil-A, phosphatidylcholine (PC) and DC-cholesterol and of colloidal systems comprised of Quil-A, cholesterol and DOTAP was studied by transmission electron microscopy (TEM). Lipid-film hydration was utilised as the first method to prepare these colloidal systems. Selected compositions containing either DC-cholesterol or DOTAP were also prepared by dialysis as second method. A novel third method for preparing homogenous dispersions of classical ISCOMs was developed utilising ethanol injection. This method was also applied in an attempt to prepare cationic modifications of ISCOMs including DC-cholesterol and DOTAP. As in the colloidal systems comprising Quil-A, PC and DC-cholesterol transformations of structures were observed upon dilution with aqueous solutions, these transitions were also studied on classical ISCOMs using TEM and dynamic light scattering techniques. Loading of cationic colloidal structures composed of Quil-A, PC and DC-cholesterol was performed with the model protein antigen ovalbumin (OVA) and a model plasmid, and the resulting structures were analysed by fluorescence spectroscopy, TEM and gel electrophoresis. The immunological properties of non-loaded and OVA-loaded structures were studied in terms of their ability to activate murine bone marrow derived dendritic cells (mBMDC) as antigen presenting cells (APC) and OVA-specific CD8+ T cells in vitro. Results: Substitution of cholesterol in classical ISCOMs with DC-cholesterol resulted in the formation of cationic cage-like structures similar to the classical particles. These were observed in pseudo-ternary Quil-A:PC:DC-cholesterol systems and even in pseudo-binary Quil-A:DC-cholesterol systems prepared by lipid-film hydration. Compositions at which cage-like structures were observed included high weight proportions of DC-cholesterol (> 60%). However, samples were relatively heterogeneous, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DC-cholesterol. The ionic strength, pH and composition of the hydration buffer were demonstrated to be important variables influencing the formation of cage-like structures. Morphological changes of pre-formed cationic cage-like structures were observed upon dilution. However, classical anionic ISCOMs showed a similar behaviour. The numbers of cationic cage-like structures appeared to increase upon prolonged storage of samples. Purification of structures and longitudinal analysis of their composition suggested an increased formation of stoichiometrically defined DC-cholesterol:Quil-A:PC complexes over time, rather than a change in composition. The substitution of phospholipid in classical ISCOMs with DOTAP also resulted in heterogeneous dispersions, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DOTAP. Phase separation phenomena were proposed based on TEM observations. However, the formation of cage-like particles with a positive [zeta]-potential was not observed. Although ethanol injection was introduced as a novel method to prepare classical ISCOMs, its application did not result in more homogenous dispersions of cationic colloidal structures containing DC-cholesterol or DOTAP. Dialysis also failed to produce higher numbers of well-defined cationic particles, although using this method homogeneous, anionic ISCOM-like particles containing DOTAP were obtained. The efficient adsorption of OVA and plasmid DNA onto cationic structures containing Quil-A, PC and DC-cholesterol was demonstrated. The adsorption process was accompanied with a decrease in [zeta]-potential, aggregation of structures and changes in the ultra-structure, particularly at high protein:lipid ratios. The in vitro immunogenicity of dispersions containing Quil-A, PC and DC-cholesterol was equivalent to that of classical ISCOMs in terms of activation of mBMDC and OVA-specific CD8+ T cells, even though smaller amounts of Quillaja saponins and total lipid were co-delivered with OVA. Furthermore, the uptake of OVA by BMDC appeared to be more efficient in conjunction with the novel cationic dispersions. Conclusions: Cationic colloidal structures containing Quillaja saponins offer great potential as vaccine delivery systems. Their advantages thus far include simple and efficient adsorption of antigen, efficient uptake by APC and immunological activity in vitro. With further development, cationic carriers containing Quillaja saponins may constitute a very potent vaccine delivery platform suitable for a variety of subunit antigens, and suffice both pharmaceutical and immunological requirements.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 260-285).