Internalization and fate of HPMA copolymers and antisense-HPMA copolymer conjugates in Hep G2 cells

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Publication Type dissertation
School or College College of Pharmacy
Department Pharmaceutics & Pharmaceutical Chemistry
Author Jensen, Keith Dale
Title Internalization and fate of HPMA copolymers and antisense-HPMA copolymer conjugates in Hep G2 cells
Date 2002-08
Description To better understand the fate of macromolecules in cells and begin to alter that fate, we studied a antisense oligonucleotide designed to inhibit the hepatitis B virus. Copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were initially used as a model compound and later they were used as a delivery carrier for the oligonucleotides. The subcellular fate of fluorescently labeled polymers was monitored by confocal microscopy and fluorescent spectrophotometry in Hep G2 cells (hepatocellular carcinoma). Semiquantitative fluorometry confirmed that the targeting moiety galactose was an effective ligand for receptor-mediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost two orders of magnitude greater than that of the nontargeted copolymer. Confocal microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and was distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm (after incubation or microinjection) accumulated in the nucleus. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed. To better understand the subcellular fate of oligonucleotides and HPMA copolymer-oligonucleotide conjugates, we studied their internalization and subcellular trafficking. A fraction of the free oligonucleotides were internalized, escaped into the cytoplasm and nucleus of Hep G2 cells, but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via non-degradable GG spacers resulted in sequestering the oligonucleotide in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a degradable GFLG spacer resulted in the release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.
Type Text
Publisher University of Utah
Subject Drug Targeting; Polymeric Drug Delivery Systems
Subject MESH Hepatitis B; Polymers; Drug Therapy; Pharmacology; Drug Delivery Systems
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "The internalization and fate of HPMA copolymers and antisense-HPMA copolymer conjugates in Hep G2 cells." Spencer S. Eccles Health Sciences Library. Print version of "The internalization and fate of HPMA copolymers and antisense-HPMA copolymer conjugates in Hep G2 cells." available at J. Willard Marriott Library Special Collection. RS43.5 2002 .J45.
Rights Management © Keith Dale Jensen.
Format Medium application/pdf
Format Extent 6,612,153 bytes
Identifier undthes,4779
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Funding/Fellowship NIH grants CA1578 and CA88047 from the National Cancer Institute, NIH training grant GM08537 and Fellowship from the American Foundation of Pharmaceutical Education.
Master File Extent 6,612,226 bytes
ARK ark:/87278/s6416zs8
Setname ir_etd
Date Created 2012-04-24
Date Modified 2012-04-24
ID 190605
Reference URL