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

This thesis describes the isolation, immobilisation and permeabilisation of <i>Digitalis lanata</i> (foxglove) and <i>Nicotiana tabacum</i> (tobacco) plant cells and protoplasts. Protoplasts were isolated from leaves of <i>Digitalis lanata</i> and <i>Nicotiana tabacum</i> and cultured in conditions of varying osmotic potential, illumination and cell density in order to achieve maximum cell stability. The regeneration of cellulose cell walls in <i>Nicotiana tabacum</i> protoplasts could be inhibited by the addition of dilute concentrations of the herbicide 2,6-Dichlorobenzonitrile. At concentrations of 2-10 mg/l in culture medium cell wall regeneration could be prevented for up to 6-8 weeks. Isolated cells and protoplasts were stabilised by entrapment within a beaded agarose support matrix. <i>Nicotiana tabacum</i> protoplasts and cells, immobilised within an agarose beaded support matrix were used to develop a technique to permeabilise and destabilise the cell membranes. Cells or protoplasts pre-loaded with 6-Carboxy-Fluorescein, Neutral Red stains and <SUP>14</SUP>C-Sucrose or <SUP>86</SUP>Rb<SUP></SUP>+ radioactive tracers were employed as markers for cell permeability or leakiness. Efflux or Compartmental analysis was used to determine the influence of various selected permeabilising agents on the integrity and the leakiness of either protoplast or cell membranes. Immobilised protoplasts or cells were subjected to a three - step procedure involving initial loading with <SUP>86</SUP>Rb<SUP></SUP>+ tracer, a membrane permeabilisation step and finally a recovery step. Protoplast membrane stability could not be regained after the recovery step, following a 30 minute period of permeabilisation with 50 mM acetic acid in culture medium. Immobilised cells could, however, regain membrane integrity with good intracellular retention of <SUP>86</SUP>Rb<SUP></SUP>+ tracer ion during the efflux experiment. Thus, immobilised <i>Nicotiana tabacum</i> cells could be made reversibly permeable with the use of specific agents. It is anticipated that such techniques which encourage reversible permeabilisation of immobilised cells have potential in larger scale plant cell culture systems to effect the release of intracellularly stored, useful secondary metabolites. There are also possibilities for the inclusion of exogenous precursors, cofactors and foreign genetic material which would increase compound yield and may produce novel secondary metabolites.

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