Since its discovery in 1998, RNA interference (RNAi) has rapidly become a routine and powerful tool for use in basic research and has also gained momentum in development as a therapeutic [1
]. While RNAi is an elegant, endogenous and conserved mechanism to selectively silence genes, inefficient delivery of exogenous short interfering RNA (siRNA) molecules to cells and tissues remains a barrier to its therapeutic development. As a result, the design of effective siRNA delivery systems is crucial for the clinical advancement of RNAi. Specifically, delivery vectors must be designed to effectively complex with nucleic acid molecules and aid in overcoming intracellular barriers such as endosomal escape and cytoplasmic vector dissociation.
A variety of molecules including polymers, lipids, and peptides have been studied for their effectiveness as delivery vectors for DNA and RNA molecules [4
]. Successful delivery vectors must exhibit a combination of functional attributes. Polymeric carrier molecules should be cationic to complex with nucleic acids, possess a high buffering capacity, exhibit low cytotoxicity, and also contain chemically reactive groups that can be modified for the addition of targeting moieties or other groups [4
Highly branched, dendritic polymers including poly(amidoamine) (PAMAM) have recently attracted interest as nucleic acid delivery vectors. Previous work has demonstrated that dendrimers can bind to DNA and RNA molecules and mediate modest cellular delivery of these nucleic acids [6
]. Recently, some studies have evaluated the use of PAMAM dendrimers for successful delivery of siRNA or antisense molecules. Generation 5 dendrimers were found to have poor cellular delivery of siRNA to NIH 3T3 MDR cells, though they mediated moderately effective delivery of antisense oligonucleotides [10
]. Another study found that increasing the PAMAM dendrimer generation to seven to increase the number of primary amine groups significantly enhanced siRNA delivery efficiency, possibly by enhanced amine-induced pH buffering[11
]. However, cytotoxicity of highly cationic dendrimers is a marked problem that hinders their widespread use in drug and gene delivery [6
Thus, it is desirable to exploit the potential of PAMAM dendrimers for nucleic acid delivery applications while reducing their cytotoxicity. The cytotoxicity of dendrimers can be reduced by conjugating hydrophilic polymers to the periphery of the dendrimer [12
], by conversion of a fraction of the cationic amine groups to uncharged moieties [15
], or by modifying a neutrally charged dendrimer with a few cationic amino acid groups sufficient to facilitate nucleic acid complexation[18
]. Since a wide variety of modifications exist to alter the properties of dendrimers, the development of structure-activity relationships will accelerate determination of the optimal dendrimer properties for a particular application [19
In this study, the effect of primary amine acetylation of the cationic, dendrimeric polymer, PAMAM, on siRNA delivery was analyzed. Previous studies have shown that neutralizing charges on the cationic polymer, polyethylenimine (PEI), by amine acetylation enhanced the transfection efficiency of plasmid DNA [22
]. This marked improvement in transfection efficiency correlated with decreased polymer/DNA interactions, thus promoting intracellular unpackaging of DNA from the polymer [23
]. Furthermore, amine acetylation of cationic polymers is attractive since it has been shown to reduce cytotoxicity in a variety of different cell lines [16
]. We studied dendrimer/siRNA interactions as well as the ability of acetylated dendrimers to deliver siRNA to cells and elicit a gene silencing effect. In addition, we evaluated the tradeoff between reduced polymer/siRNA interactions and reduced endosomal buffering capacity. These design parameters are important in the rational modification of PAMAM dendrimers for siRNA delivery.