RESUMEN
Herein, we demonstrate the reversible addition-fragmentation chain transfer (RAFT) synthesis of an adamantane-conjugated glycopolymer, poly(2-methacrylamido-2-deoxy glucopyranose) (Ad-pMAG), as a hydrophilic coating to promote colloidal stability of click cluster-pDNA complexes in biological media. The Ad-pMAG is assembled via noncovalent interactions through inclusion complex formation between adamantane (Ad) and the ß-cyclodextrin (ßCD) core of the click cluster/pDNA and then further assembled with plasmid DNA to form polyplexes. Ad-pMAG incorporation was favorable over Ad-poly(ethylene glycol) (Ad-PEG) due to the enhanced colloidal stability of the click cluster/pDNA polyplex under physiological salt conditions at high N/P ratios. Interestingly, the uptake and reporter gene expression with polyplexes coated with the Ad-pMAG was much lower in HeLa cells than that observed with two glioma cell lines (U87 and U251 cells) in vitro, possibly indicating some delivery specificity.
RESUMEN
Understanding the mechanisms of cellular internalization is necessary for rational design of efficient polymers for DNA delivery. In this paper, we present evidence that poly(glycoamidoamine) (PGAA)-DNA complexes (polyplexes) interact with cell-surface glycosaminoglycans (GAGs) in a manner that is not solely dependent on charge. The presence of GAGs appears to be necessary for efficient cellular uptake, as polyplex internalization was decreased in GAG-deficient CHO (pgsA-745) cells. However, uptake was nearly unaffected in cells deficient only in heparan sulfate. Internalization of PGAA polyplexes appears to be dependent on GAG sulfation in mammalian cell lines, yet the PGAA polymers are decomplexed from pDNA by high concentrations of GAGs in a charge-independent manner. This finding suggests that interactions between the carbohydrates on the polymer and GAGs may contribute to polyplex binding. Quartz crystal microbalance studies support the findings that relative PGAA polyplex-GAG binding affinities are also not completely mediated by charge. As measured by dynamic light scattering and TEM, GAGs appear to accumulate on the surface of polyplexes without disrupting them at a lower concentration, which may stimulate cellular internalization due to close interactions between the polyplexes and the GAGs. Gel electrophoresis and fluorescence measurements of an intercalating dye suggest that polyplex interaction with GAGs can induce dissociation, which could represent a potential pDNA release mechanism. These results imply that similar interactions may occur on cell surfaces, and strongly supports the hypothesis that GAGs function as cell surface receptors for polyplexes formed with PGAA vehicles.