Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Abstract

Background: Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intra cellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silenc ing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methylβcyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolindependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methylβcyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrindependent and caveolindependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrindependent and caveolindependent endocytosis vs only caveolindependent endocytosis in T98G cells.

Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex

“…LysoTracker ® is a highly specific, one-step staining dye for acidic organelles and widely employed to track lysosomes. 23 As the merged images illustrated (Figure 1A), only a very small amount of the siRNAs were entrapped in late-endosomes/lysosomes between 0.5 and 6 h post-transfection. Moreover, majority of the siRNAs were localized in the cytoplasm and few translocated into the nucleus at 3 and 6 h post-transfection.…”

Anti-Prion Drug mPPIg5 Inhibits PrPC Conversion to PrPSc

“…However when examined individually, each of the arguments quickly becomes less robust. For example, chloroquine has multiple effects on cells in addition to raising its lysosomal pH such as altering protein trafficking and endocytosis [31], [32]. In addition, NH 4 Cl, another alkalising lysomotrophic agent, does not inhibit the effect of PEI dendrimers [6] suggesting alkalisation of acidic compartments alone is not sufficient.…”

<p>PAMAM Dendrimer Nanomolecules Utilized as Drug Delivery Systems for Potential Treatment of Glioblastoma: A Systematic Review</p>

“…For instance, Perez and colleagues (2011) experimented with a larger G7 dendrimer complex which carried siRNA to T98G (GB) and J774 (macrophage) cells. 34 Dendrimers of this size are seldom used as newer investigations have found that smaller dendrimers, such as G4, are more effective in crossing the BBB while as effective in delivering the cargo. Nonetheless, Perez and colleagues (2011) found that in T98G cells, the following treatments and respective resulting measures of reduction in glioblastoma cell proliferation were observed: MbetaCD -75%, genistein -50%, and chloroquine -10-20%.…”

“…29 them. 34 The dendrimer core also influences the space (ie, "void") between dendrons. For example, compared to a more standard ammonia core, a triethanolamine (TEA) core has several flexible ethylene glycol units, which expand the distance between dendrons, creating a larger void and enhancing the delivery of genes.…”