Translocation of macromolecules into whole rat lenses in culture.
OBJECTIVE
Little is known about the endocytosis and transcytosis of macromolecules into lens epithelium and fiber cells. The objective of this study was to determine if proteins (alpha-crystallins, beta-crystallins, and gamma-crystallins), carbohydrate (dextran), and plasmid DNA translocate from culture medium into these parts of the lens, with and without prior encapsulation into liposomes.


METHODS
alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were coupled with the fluorochrome Texas red, and plasmid DNA was labeled with propidium iodide. Adult rat lenses were incubated in medium containing one of these components with and without prior encapsulation of the macromolecule in commercially available liposomes (BioPORTER for alpha-crystallins, beta-crystallins, gamma-crystallins, and dextran; GenePORTER for plasmid DNA). Translocation of fluorescent macromolecule from the medium into the lens capsule, epithelium and fiber cells was monitored by confocal microscopy.


RESULTS
alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were present in the capsule, epithelium, and fiber cells after 5 h of incubation. Translocation of fluorescent protein macromolecules into the epithelium was greatly facilitated by encapsulation in BioPORTER liposomes. These macromolecules were localized within the cytoplasm of epithelium and fiber cells. Plasmid DNA was localized to the epithelium, but not the fiber cells. Prior encapsulation of plasmid DNA into GenePORTER liposomes did not increase the intensity of fluorescence localized in epithelium. Without encapsulation, plasmid DNA preferentially localized to the nuclei of epithelial cells, while after encapsulation, plasmid DNA preferentially localized to the cytoplasm.


CONCLUSIONS
After incubation with cultured lenses, large macromolecules comprised of proteins and carbohydrates were localized within the cytoplasm of epithelial cells and fiber cells. Prior encapsulation of protein macromolecules into BioPORTER liposomes facilitated the translocation of macromolecules into the cytoplasm of epithelium. Incubation of lenses with plasmid DNA resulted in localization to the epithelium, but not fiber cells. Localization of plasmid DNA was not facilitated by prior encapsulation in GenePORTER. Encapsulated DNA preferentially localized to the cytoplasm of epithelial cells, while without encapsulation, plasmid DNA localizes to the nuclei of epithelial cells. Together, these studies demonstrate that macromolecules of potential biological importance can readily pass through the lens capsule into epithelial cells and in some cases transcytose through the epithelium into fiber cells of the cortex. Furthermore, these studies suggest that prior encapsulation of protein macromolecules may be a possible therapeutic delivery system of physiologically important macromolecules into the epithelium and/or fiber cells of the intact lens.
Division of Biology, Kansas State University, Manhattan, KS 66506, USA. dboyle@ksu.edu.
Daniel L
DL
Boyle
Paul
P
Carman
Larry
L
Takemoto
D000818:Animals; D003459:Crystallins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003911:Dextrans / Q000378:metabolism*; D004337:Drug Carriers; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism; D005456:Fluorescent Dyes; D007903:Lens Capsule, Crystalline / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D008081:Liposomes; D046911:Macromolecular Substances; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D014966:Xanthenes
D003459:Crystallins; D003911:Dextrans; D004337:Drug Carriers; D005456:Fluorescent Dyes; D008081:Liposomes; D046911:Macromolecular Substances; D014966:Xanthenes; C034657:Texas red; D004247:DNA