Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium
Vercauteren, D. and Piest, M. and Soraj, M. Al and Jones, A.T. and Engbersen, J.F.J. and Smedt, S.C. de and Braeckmans, K. (2010) Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium. Journal of Controlled Release, 148 (1). e99-e100. ISSN 0168-3659
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|Abstract:||In vitro endocytosis of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE) was studied, the latter being an interesting target for ocular gene therapy. We found that cationic CBA ABOL DNA polyplexes attach to cell surface proteoglycans of these RPE cells and get subsequently internalized via a phagocytosis-like mechanism, as well as Flotillin dependent endocytosis.
Proper delivery of therapeutic genes to designated cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. To this end, typically sub-micron sized particles are made by combining the therapeutic genes with a carrier material, such as cationic polymers, that aid in delivering the genes to the target site. In this work, for the first time, we have evaluated the ability of the highly promising bioreducible polymer carrier cystamin bisacrylamid aminobutanol (CBA ABOL)  (Fig. 1) to deliver plasmid DNA in cultured cells of the retinal pigment epithelium (ARPE-19) and characterized in vitro the cellular interactions with these target cells. These studies are of crucial importance since the further design and functionalization of polymeric gene carriers depend strongly on our understanding of the mechanisms involved in cellular adhesion, intracellular uptake and intracellular processing of the polyplexes.
ARPE-19 cells (retinal pigment epithelial cell line; ATCC number CRL-2302) were cultured in DMEM:F12 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 2% penicillin-streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. CBA ABOL gene complexes with an average hydrodynamic diameter of 130 nm and an average zeta potential of + 45 mV in 20 mM HEPES buffer were obtained by adding the polymer in a mass ratio of 48/1 in 20 mM HEPES to the plasmid and vortexing the mixture for 10 min. For every transfection, fresh polyplexes were prepared and applied to the cells within 30 min after complexation. For all uptake studies, YOYO-1™ (λex = 491 nm, λem = 509 nm, Molecular Probes, Merelbeke, Belgium) labeled pGL4.13 plasmid (Promega, Leiden, The Netherlands) was used. For all transfection studies, gWiz™GFP plasmid (Aldevron, Freiburg, Germany) was used. siRNAs were all purchased from Dharmacon and transfected in cells with the help of LipofectaminRNAiMAX (Invitrogen, Merelbeke, Belgium). Protein knockdown was assessed on Western Blot. Uptake of polyplexes or endocytic markers or GFP expression was measured on a FACS Calibur Flow Cytometer (Beckton Dickinson, Erembodegem, Belgium). For genetic labeling of endosomes, cells were transfected with GFP-fusion proteins.
For fluorescence colocalization studies with GFP labeled cellular structures, cells were transfected with GFP-fusion proteins using Lipofectamin2000 (Invitrogen, Merelbeke, Belgium) and 24 h later exposed to red fluorescent labeled polyplexes. For this, CBA ABOL was complexed with pGL4.13 plasmid, covalently labeled with Cy5 (Label IT Nucleic Acid Labeling Kit, Mirus Bio Corporation, WI, USA). Live cell fluorescence colocalization was then performed on a custom built laser epi-fluorescence microscope set-up. A Nikon Plan Apo VC 100× 1.4 NA oil immersion objective lens (Nikon Belux, Brussels, Belgium) was used for imaging. GFP and Cy5 were excited with 491 nm and 636 nm laser light and emission was detected on an EMCCD camera (Roper Scientific, Nieuwegein, The Netherlands). For live cell imaging the cells were placed in a stage top incubation chamber (Tokai Hit, Shizuoka, Japan), set at 37 °C, 5% CO2 and 100% humidity.
Result and discussion
First, we found evidence that these net positively charged CBA ABOL polyplexes adhere to the negatively charged heparan sulfate proteoglycans (HSPGs) at the cell surface and that polyplex internalization is blocked by antibodies against Toll-like receptor 9
|Copyright:||© 2010, Elsevier|
Science and Technology (TNW)
|Link to this item:||http://purl.utwente.nl/publications/77532|
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