Liposomes are small phospholipid vesicles that have been widely investigated as drug carriers for the delivery of therapeutic agents. A variety of liposome formulations are presently under clinical trial exploration, while others have already been approved for clinical use. The aim of this study was to optimize liposome uptake into bacterial cells. Both gram-positive and gram-negative bacteria were used in the study as well as Candida albicans.Response surface methodology (RSM) using a central composite design (CCD) model was used to optimize liposomal formulations of carboxyfluorescien (CF) for each of the three microbes, and also the three microbes in combination namely; Staphylococcus aureus (Sa), Escherichia coli (Ec) and Candida albicans (Ca). Percentage of CF encapsulated and CF increase in Uptake were investigated with respect to two independent variables that were, cholesterol (CHOL) and stearylamine (SA) content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each microbe and the three microbes in combination. The model selected by the software managed to reasonably correlate the predicted models to the experimental data. Encapsulation of carboxyfluorescien (CF) into a liposome formulation enhanced its uptake by Staphylococcus aureus and Escherichia coli as well as Candida albicans. This was evident in the increase in CF uptake when the uptake rate of free CF was compared with that of liposomal CF.

Thesis (M.S.)--University of Toledo, 2010. / Typescript. "Submitted to the Graduate Faculty as partial fulfillment of the requirements of the Master of Science degree in Pharmaceutical Sciences with Industrial Pharmacy Option." "A thesis entitled"--at head of title. Title from title page of PDF document. Bibliography: p. 87-102.

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.

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.

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.

abstract / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.