Studying the Fate of Antisense Oligonucleotide/Polymer Conjugates Targeted to Hep G2 Cells.
Keith D. Jensen, Pavla Kopeckova, Jindrich Kopecek.
14th Annual Meeting of the American Association of Pharmaceutical Scientists, New Orleans, Louisiana, November 14-18, 1999, AAPS PharmSci ( 1 (4) Supplement. Abstract #4099.


Purpose. To study the internalization, sub-cellular trafficking and ultimate fate of antisense oligonucleotides in Hep G2 cells. These data are a prerequisite for the development of a polymer based oligonucleotide delivery system.

Methods. A targeted water soluble polymer is used to delivery the oligonucleotide to the cells. The delivery system is comprised of a copolymer of N-(2-hydroxypropyl)-methacrylamide to which the oligonucleotide and galactosamine (for targeting) were covalently attached. The oligonucleotide used was a fluorescein labeled 21-mer phosphorothioate oligodeoxynucleotide. The delivery system was tested in vitro using Hep G2 cells. Cell associated fluorescence was quantified on a fluorometer. Confocal laser microscopy was used to monitor the internalization and fate inside the cells.

Methods. The fate of the delivery system was first tested by incubating cells with fluorescein labeled polymer (no oligonucleotide attached). Previously reported results showed that galactose efficiently targets the polymer to Hep G2 cells. Using confocal fluorescent microscopy, we found that the polymer quickly entered the cells and initially remained in small endososmal/lysosomal vesicles. After incubation with higher polymer concentrations, we observed that polymer began to escape into the cytoplasm and nucleus of a small fraction of the cells within 24 hours. Within 72 hours, a large fraction of the cells had polymer in their cytoplasm and nucleus. Entry into the cytoplasm and nucleus was unexpected and the mechanism of transport is currently unknown. To help elucidate the fate of the polymer, microinjection was employed. Preliminary results found that polymer injected into the cytoplasm began to enter the nucleus in less than 40 minutes post-injection. Polymer injected into the nucleus tended to remain there up to 48 hours followed by entry into the cytoplasm. These studies were complicated by a difference in response between cells in the sample set. We are currently optimizing a system to study live cells to help assess the fate in one set of cells over time. Studies with oligonucleotide/polymer conjugates are underway.

Conclussions. We found that confocal microscopy can detect and distinguish fluorescein labeled polymer in small vesicles, the cytoplasm and nucleus and we were able to follow it through these various compartments. Many cells exhibit nuclear localization of the polymer (with little in the cytoplasm) after cytoplasmic or nuclear injection. Nuclear accumulation is also observed after incubation, although the majority of polymer remains in small vesicles. We do not yet know the reason for nuclear localization.

KDJ was supported by an AFPE fellowship and NIH Grant GM08573.

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