RNA interference utilizes short-interfering RNAs (siRNAs) to selectively silence a gene and, when targeted towards an oncogene, has potential for use as an anticancer therapy. The advancement of RNAi-based therapies is limited by the lack of effective siRNA delivery vectors. The use of cationic polymers as siRNA delivery vectors is promising, as synthetic polymers can be easily functionalized or modified for a particular application. Rational design of polymeric carriers, though, has been complicated by the fact that most chemical modifications affect multiple aspects of the delivery process.

In this work, polyamidoamine (PAMAM) dendrimers are being studied as vectors for the targeted delivery of siRNAs to U87 malignant glioma cells. The highly organized, branched structure of PAMAM makes it an attractive polymer for targeted drug delivery due to favorable ligand presentation and the presence of many reactive amine groups. The extent of primary amine acetylation of generation 5 poly(amidoamine) (PAMAM) dendrimers was studied as a modification for the delivery of siRNA to U87 malignant glioma cells. A linear decrease in cytotoxicity was observed upon primary amine acetylation. To evaluate the siRNA delivery by acetylated PAMAM and subsequent gene silencing dynamics, U87 cells stably expressing d1EGFP were analyzed using flow cytometry. We observed that a modest fraction (approximately 20%) of primary amines can be modified while maintaining the GFP silencing ability comparable to unmodified PAMAM, but higher degrees of amine neutralization notably reduced the gene silencing efficiency of PAMAM-siRNA polyplexes. This trend might be explained by a marked reduction in endosomal buffering capacity of dendrimers upon amine acetylation, which counteracted the increase in siRNA unpackaging.

To improve the siRNA delivery of dendrimers to tumor cells, α_vβ₃ integrin-targeting RGD peptides have been conjugated to PAMAM dendrimers using a sulfo-LC-SPDP crosslinker. An increase in intracellular delivery of fluorescently-labeled siRNA to U87 cells that highly express the α_vβ₃ integrin receptor was observed upon incorporation of RGD targeting peptides into the delivery system. Enhanced delivery was not observed, however, when RGD-targeted dendrimers were delivered to A172 malignant glioma cells that do not express α_vβ₃ integrin receptors. This indicates that the uptake of RGD-conjugated dendrimers occurs at least in part through the α_vβ₃ receptor mediated mechanism.

Currently, we are evaluating the effect of varying extents of RGD-conjugation to PAMAM dendrimers on cellular delivery of siRNA. We expect that increasing the extent of RGD conjugation will have a synergistic effect on cellular binding and subsequent siRNA delivery due to multivalent interactions between targeted dendrimers and cell-surface α_vβ₃ receptors. However, a dramatic change of the dendrimer chemistry might alter its intracellular trafficking and have a detrimental effect on its ability to deliver siRNA. Therefore, surface plasmon resonance will be used to quantatively study the interactions between targeted dendrimers with α_vβ₃ integrin proteins, and the dendrimer chemistry (extent of acetylation and degree of peptide conjugation) will be optimized to achieve a maximum biological response.