Efficient in Vitro siRNA Delivery and Intramuscular Gene Silencing Using PEG-Modified PAMAM Dendrimers

Abstract

Although siRNA techniques have been broadly applied as a tool for gene knockdown, substantial challenges remain in achieving efficient delivery and in vivo efficacy. In particular, the low efficiency of target gene silencing in vivo is a critical limiting step to the clinical application of siRNA therapies. Poly(amidoamine) (PAMAM) dendrimers are widely used as carriers for drug and gene delivery; however, in vivo siRNA delivery by PAMAM dendrimers remains to be carefully investigated. In this study, the effectiveness of G5 and G6 PAMAM dendrimers with 8% of their surface amines conjugated to MPEG-5000 was studied for siRNA delivery in vitro and for intramuscular in vivo delivery in mice. The results from the PEG-modified dendrimers were compared to the parent dendrimers as well as Lipofectamine 2000 and INTERFERin. Both PEG-modifed dendrimers protect the siRNA from being digested by RNase and gave high transfection efficiency for FITC-labeled siRNA in the primary vascular smooth muscle cells (VSMC) and mouse peritoneal macrophages. The PEG-modified dendrimers achieved knockdown of both plasmid (293A cells) and adenovirus-mediated green fluorescence protein (GFP) expression (Cos7 cells) in vitro with efficiency similar to that shown for Lipofectamine 2000. We further demonstrated in vivo that intramuscular delivery of GFP-siRNA using PEG-modified dendrimer significantly suppressed GFP expression in both transiently adenovirus infected C57BL/6 mice and in GFP transgenic mice.

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

“…Studies of the ability of siRNA with transfection reagents [e.g., cationic liposomes (1), polyelectrolyte micelles (2), and dendrimers (3)] to reduce the expression of genes in cell culture often take advantage of the addition of siRNA and transfection reagents to cells in media that are free of serum for a period of time [e.g., 3-24 h before replacement with media supplemented with 10% (vol/vol) FBS]. Such a procedure provides a window of time for the entry of siRNA into cells that avoids exposure of the siRNA to serum nucleases that would otherwise catalyze the hydrolysis of siRNA; such a procedure also leaves the challenge of short half-lives in serum-containing biological media unaddressed.…”

Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery

“…Encapsulation of siRNAs within nanoparticles offers numerous delivery benefits, including protection from degradation by ubiquitous nucleases, passive and active targeting, and evasion of endosomal Toll-like receptors (1)(2)(3). To date, several polymeric, lipid, and dendritic nanoparticles have been developed for the encapsulation and delivery of siRNAs (4)(5)(6)(7)(8)(9). Despite the delivery successes met by some of these carriers, advances are necessary to allow the fullest application of siRNA in the clinic.…”

Non-Viral Delivery and Therapeutic Application of Small Interfering RNAs

“…Intramuscular administration of these complexes to GFP-transgenic mice also revealed a decrease in the expression level of mRN A of the green fluorescent protein. PAMAM nanoparticles were shown to reliably protect siRN As against blood serum nucleases [95]. …”