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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27395 |
| Date | 17 October 1980 |
| Creators | Parker, Robert S. |
| Contributors | Selivonchick, D. P. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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