Thesis (Ph. D.)--University of Wisconsin--Madison, 1980. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 227-256).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1980. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

No description available.

Receptor mediated endocytosis allows for the site specific delivery of exogenous DNA via appropriate ligand-receptor interactions. Various ligands have been used to target the asialoglycoprotein receptor (ASGP-R) present on the hepatocyte cell membrane viz. asialofeutin, asialoorosomucoid, lac-BSA, asialolactoferrin, asialotransferrin, asialo-ceruloplasmin and galactose. The high affinity that the receptor displays for the galactose sugar moiety has led to the development of several new galacto-lipids for the incorporation into liposomes intended for hepatocyte targeting. In this study, three cholesteryl derivatives displaying galactose units linked to the sterol skeleton by different spacer elements have been formulated into cationic liposomes with and without polyethylene glycol (PEG) accessories. The three galactosylated liposomal formulations were prepared using near equimolar amounts of MSO9 (N,N-dimethylaminopropylamidosuccinyl-cholesterylformylhydrazide) and DOPE (dioleoylphosphotidylethanolamine) together with the respective galactose derivative (at 10 mole % w/w) viz. Cholesteryl-3β-N-(4-aminophenyl-β-Dgalactopyranosyl) carbamate; Cholesteryl (1-β-D-galactopyranosyl-1,2,3 triazol-4-yl) carbonate; and Cholesteryl-β-D-galactopyranoside. All liposomes displayed DNA binding, nuclease protective capabilities to plasmid DNA, low cytotoxicity (cell viability being within 60-101 %) and an increase in transfection activities, in the human hepatocellular carcinoma cell line HepG2, which expresses the ASGP-R abundantly. The results obtained correlate well with differences in the spacer element in the 3 galactosylated cholesterol derivatives under study and the presence and absence of 2 mole % DSPE-PEG₂₀₀₀ in the liposome formulations. Overall, it was observed that the cationic liposome containing cholesteryl (1-β-Dgalactopyranosyl- 1,2,3 triazol-4-yl) carbonate (with and without PEGylated accessories), which was synthesised chemically using “click chemistry”, afforded the highest in vitro transfection activity, and may be optimised and studied further. The highest levels of transfection activity, in vitro, were attributed to the increased length of the spacer arm between the galactose moiety and the cholesteryl anchor of the targeting component. Two formulations were then subjected to in vivo studies, using male Sprague Dawley rats which yielded little or no transgene expression. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

The ingestion, uptake, and metabolism of liposomes by juvenile Pacific oysters (Crassostrea gigas) were studied by several methods in an effort to assess their potential as encapsulating agents. Liposomes composed of egg phosphatidylcholine-cholesterol-stearylamine (7:1:2) formed readily and appeared stable in 20°/oo seawater. Radiotracer studies with liposomes made with ¹⁴C-labeled cholesterol or phosphatidylcholine showed uptake of up to 40% of the dose in 24 hrs, with the majority of uptake occurring in the visceral mass. Only slight amounts of label were observed in adductor muscle or mantle tissue. Absence of label in free fatty acids in oysters fed liposomes made with di[l-¹⁴C] palmitoyl phosphatidylcholine indicated a lack of significant amounts of fatty acid hydrolysis from phospholipid in the stomach or lumen of the digestive diverticula. However, radioactivity was observed in lipid other than phosphatidylcholine, including triglyceride, phosphatidylethanolamine, and an unidentified polar lipid. Radioactivity in these lipids resided exclusively in the fatty acids, indicating breakdown of the ¹⁴C-phosphatidylcholine via acyl transfer. To examine metabolism of liposome-encapsulated substances, [1-¹⁴C]glucose and [U-¹⁴C]amino acids were entrapped and fed to oysters. Label from glucose appeared largely in a choloroform-methanol-insoluble fraction, with little radioactivity recovered in the lipid or soluble aqueous fractions. Most label from amino acids was recovered in trichloroacetic acid-precipitable protein. Control oysters given the same amounts of non-encapsulated [1-¹⁴C] glucose or [U-¹⁴C]amino acids as in liposome trials showed (1) the same uptake of label from free amino acids in comparison with encapsulated glucose, and (2) increased uptake of free, amino acids in comparison with encapsulated amino acids. Label from free glucose or amino acids entered the same fractions as encapsulated label. Evidence for intracellular uptake of liposomes was obtained with fluorescence microscopy after feeding oysters with liposomes containing bovine serum albumin conjugated with fluorescein isothiocyante (FITC). The appearance of small fluorescent inclusions within the apical portions of many of the ducts and tubules of the digestive diverticula suggest phagocytosis of intact liposomes. Uptake was not observed in other parts of the alimentary canal. The feeding of liposomes in which the stearylamine had been conjugated with FITC resulted in generalized fluorescence in most of the digestive diverticula and stomach epithelium, perhaps due to extracellular hydrolysis of FITC and its subsequent diffusion into epithelial cells. No fluorescence occurred in tissues other than those of the digestive tract. Autoradiography studies with liposomes containing di[l-¹⁴C]palmitoyl phosphatidylcholine showed radioactivity dispersed throughout the epithelial cells of the ducts and tubules of the digestive diverticula. Only slight radioactivity was observed in the intertubular connective tissue or the lumen of the tubules or stomach. This distribution of liposomal materials resembled that of fluorescence from feeding trials with FITC-tagged liposomes, and indicated uptake of intact liposomes followed by intracellular breakdown and dispersal of the liposomal components. To investigate the process of particle selection in oysters, polyacrylamide beads (2 [plus or minus] 1μ) with aminoethyl side groups, and beads with FITC-conjugated side groups were fed to oysters. Large quantities of both types of beads were observed in the stomach and intestine, but not in the digestive diverticula, indicating recognition as non-food particles despite their organic nature. The ingestion of such derivitizable particles suggests their use in studies of acceptance-rejection processes in the stomach of bivalves. The ingestion, intracellular uptake, and breakdown of liposomes and their contents indicates a use for these particles in studies of nutrition or pollutant-food web relationships in bivalve molluscs or other filter-feeding organisms. / Graduation date: 1981

No description available.

No description available.

No description available.

Immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through the triggering of the endosomally localized TLR9 by a poorly understood mechanism. The immunopotency and broad range of activity of CpG ODN makes it a promising immunotherapeutic for the treatment and prevention of cancer and other diseases. However, rapid degradation of ODN by serum nucleases, low levels of accumulation in target tissue and lack of specificity for and poor uptake into target cells following systemic administration pose significant hurdles for the clinical application of CpG ODN. This thesis describes the immunostimulatory properties of CpG ODN encapsulated in liposomal nanoparticles (LN), a delivery system that overcomes many of the problems impeding the clinical development of "free" ODN. In particular, it is shown that LN delivery of CpG ODN specifically targets the ODN for uptake by immune cells in vivo, providing a basis for significantly enhanced immunostimulatory activity, including more potent innate and adaptive immune responses, that ultimately improve anti-tumour efficacy. A particular focus of this thesis concerns previous observations that methylated sequences in ODN (mCpG ODN) are immunologically inert. It is shown that encapsulation of mCpG ODN in LN results in immunostimulatory activity that is equal to or greater than that observed for LN formulations of the equivalent unmethylated form as judged by various immune parameters and anti-tumour efficacy. Further, it is shown that both LN-mCpG ODN and LN-CpG ODN exert their immunostimulatory effects via TLR9 based on preliminary in vitro results and confirmed by studies performed in TLR9 knockout animals. The mechanisms responsible for the differentiation between both CpG ODN and mCpGODN and how encapsulation endows immunostimulatory potential are explored. It is shown that discrimination occurs upstream of TLR9 and that the lack of immunological activity of free mCpG ODN is not due to differences in uptake, trafficking to endosomal compartments or ability to bind to TLR9, when compared with CpG ODN, but rather due to its ability to colocalize with TLR9 in the endosomal compartment. It is proposed that whereas the uptake of free CpG ODN results in the induction of the Src Family Kinase signalling cascade which mediates the migration of TLR9 from the ER to the late endosome, the uptake of free mCpGODN does not. However, it is suggested that encapsulation bypasses the methylation specific recognition of CpG ODN, allowing for the activation of SFK signalling resulting in subsequent co-localization of TLR9 and mCpG ODN in the endosome thus initiating immunostimulatory activity.