(208f) PAMAM-RGD Conjugates for siRNA Delivery through a Three-Dimensional Tumor Model of Malignant Glioma

In this work, the ability of PAMAM-RGD conjugates to mediate siRNA delivery to U87 glioma cells cultured both in two- and three-dimensions was evaluated. Generation 5 PAMAM dendrimers were cross-linked with a RGD integrin-targeting peptide sequence, cyclo(RGDfC). The ability of PAMAM-RGD conjugates to inhibit α_vβ₃ integrin-mediated adhesion of U87 cells to the extracellular matrix protein, fibrinogen, was confirmed using a competitive-binding cell adhesion assay.

A three-dimensional cell culture model of malignant glioma was developed to evaluate the ability of PAMAM-RGD conjugates to facilitate siRNA delivery in three-dimensions. Spheroids of U87 cells were grown using the hanging drop method. Spheroids were treated with dendrimer/siRNA complexes containing a fluorescently-labeled siRNA sequence. Confocal microscopy was used to visualize siRNA delivery throughout the spheroid volume. siRNA delivery mediated by dendrimers not containing a RGD targeting sequence was confined to the periphery of spheroids, indicating poor penetration through the multicellular structure. On the contrary, in spheroids treated with PAMAM-RGD/siRNA complexes, siRNA delivery was observed throughout the spheroid volume, indicating enhanced penetration of the spheroids by the presence of RGD targeting peptides. We expect that the enhanced spheroid penetration by PAMAM-RGD conjugates is due to the ability of PAMAM-RGD to interfere with the cell-ECM interactions found in three-dimensional spheroids and are currently performing experiments to test this hypothesis.