Hepatocyte-directed liposomal gene delivery systems have received much attention in view of the present lack of suitable treatment alternatives for several liver-associated disorders. While targeting of liposomes to the asialoglycoprotein receptor (ASGP-R), nearly-exclusive to hepatocytes, is a well-documented means of achieving cell-specificity, several intra- and extracellular barriers reduce the efficacy of liposomal gene transfer. These include the aggregation and opsonisation of lipoplexes by serum components; and endo/lysosomal degradation of internalised DNA. This study has attempted to address the individual concerns by modifying hepatotropic liposomes with a steric stabilising, polyethylene glycol (PEG) shroud, and an endosomal escape-inducing proton sponge moiety. Novel galactosylated (SH02) and imidazolylated (SH04) cholesterol derivatives were successfully synthesised with the aim of conferring the respective functions of ASGP-R-specificity and proton sponge capability upon cationic liposome formulations. The individual derivatives afforded stable, unilamellar vesicles (< 200 nm, Z-average diameter) when incorporated at 10 % on a molar basis with the cytofectin, 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and co-lipid, dioleoylphosphatidylethanolamine (DOPE). Modification of these formulations with 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000] (DSPEPEG₂₀₀₀), at 5 mol %, gave smaller vesicles (< 110 nm, Z-average diameter) and moderately reduced the instability associated with the combination of both SH02 and SH04 in a single formulation. Individual preparations formed electrostatic complexes with pCMV-luc plasmid DNA, as demonstrated by gel retardation assays and electron microscopy. Furthermore, the liposomes afforded some protection to the DNA cargo against serum nuclease attack during a 4 hour-long exposure to foetal calf serum at 37 °C. However, the DNA-binding and protecting capabilities of the liposomes were reduced upon addition of the PEG coating. Growth inhibition assays showed that lipoplexes derived from individual formulations were well tolerated by human hepatocyte-derived, HepG2, and embryonic kidney, HEK293, cell lines. Expression of the luciferase transgene mediated by non-pegylated formulations containing SH02 was significantly higher in hepatocytes than in the ASGP-R-negative, kidney cells. Furthermore, receptor-mediated internalisation of non-pegylated, galactosylated carriers by hepatocytes was demonstrated by the gross inhibition of transfection in the presence of excess asialofetuin, a natural ligand to the ASGP-R. Liposome acid titration profiles highlighted the endosomal pH-buffering capacity afforded by SH04. However, the imidazolylated lipid enhanced the transfection activity of the non-sterically stabilised Chol-T/DOPE system, but not that of its targeted counterpart, and only with respect to HEK293 cells. Finally, pegylation reduced the transfection capability of liposomes by at least three orders of magnitude in both cell lines. The results suggest that further optimisation of liposome composition is necessary in order to achieve a liposomal system that simultaneously embodies hepatocyte-targeting, proton sponge and long-circulating properties. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

[Truncated abstract] Chronic liver injury results from many etiologies ranging from viral infection to inborn errors of metabolism. A common result of liver injury is activation of hepatic stellate cells and portal fibroblasts to myofibroblasts. In chronic injury, production of extracellular matrix by activated myofibroblasts results in liver fibrosis and ultimately cirrhosis. Kupffer cells and monocytes may play an important role in the pathogenesis of certain liver diseases. Endotoxin-responsive macrophages and recruited monocytes (CD14-positive cells) are potential sources of profibrogenic factors but their potential role in the pathogenesis of liver disease has not previously been examined. The first aim of this thesis described in chapter 3 was to evaluate the hypothesis that CD14-positive macrophages/monocytes are present in the livers of patients with hereditary haemochromatosis (HH), primary biliary cirrhosis (PBC), chronic hepatitis C (HCV) and nonalcoholic steatohepatitis (NASH) and contribute to the pathogenesis of fibrosis as evidenced by co-localization of these cells with activated myofibroblasts. Liver specimens from control subjects and those with HH, PBC, HCV and NASH were immunostained for CD14, CD68 and α-smooth muscle actin and the number of cells expressing these antigens was determined. The total number of hepatic CD68-positive cells was similar in diseased and control livers. The number of CD14-positive cells correlated with advanced fibrosis in HH, PBC, HCV but not in NASH. The number of CD14-positive cells was increased with advanced inflammatory activity in HCV. CD14-positive cells were often associated with α-smooth muscle actin-positive myofibroblasts in fibrous septa. In conclusion, many forms of human chronic liver disease demonstrate increased numbers of CD14-positive macrophages/monocytes which are associated with fibrous septa and myofibroblasts. To determine whether CD14-positive cells contribute to fibrogenesis, experimental models of liver injury were used in chapters 5 and 6. The aim of chapter 5 was to determine whether CD14-positive macrophages/monocytes are detected in a bile duct ligation model of liver injury. To accomplish this aim, a novel antibody to rat CD14 was developed as described in chapter 4. A time-course study was undertaken in rats following bile duct ligation for up to 14 days. An increase in the number of hepatic CD14-positive cells was detected early following bile duct ligation, and was associated with increased gene expression of α-smooth muscle actin and procollagen I. Thus, myofibroblastic transformation in this model was associated with increased numbers of CD14-positive cells suggesting a possible relationship between the two phenomena. In order to specifically evaluate the role of CD14 in myofibroblastic transformation, a final study in CD14 knockout (KO) mice was undertaken in chapter 6

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Examensarbete.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online as viewed 10/12/2009 through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 116-130).

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 111-122). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Western Australia, 2007. / Also available online.

Thesis (Ph.D. in Toxicology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 141-156). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;