Abstract
Currently, RNAi based approaches for cancer treatment involving short double stranded RNA molecules (siRNA) are under vigorous scrutinization. Due to numerous biological obstacles, siRNA delivery into target cells requires protective escort. On the other hand, combining of siRNA-mediated gene silencing and action of conventional chemotherapeutics can propose additional enhancement of anticancer activity. In the present study, we investigated a siRNA cocktail able to downregulate anti-apoptotic genes (BCL-xL, BCL-2, MCL-1) and the chemotherapeutic agent 5-fluorouracil (5-FU) to evaluate multi-target cytotoxic effect on human cervical carcinoma cells (HeLa cell line). Novel phosphorus containing dendrimers of 3rd and 4th generations (namely AE2G3 and AE2G4) with voluminous piperidine terminal cationic groups were designed and tested as siRNA carriers. Dendrimers of both generations showed remarkable ability to bind pro-apoptotic siRNAs and provided 80–100% siRNA uptake by HeLa cells in the serum containing medium, while the widespread transfection agent Lipofectamine showed only ~40% uptake. SiRNA cocktail (in low concentrations 50 and 100 nM) delivered by AE2G3 dendrimer caused almost complete elimination of cancer cells. We have discovered considerable increase of 5-FU cytotoxic effect by addition of AE2G3/siRNA cocktail complexes in low doses. Thus, we demonstrated the effectiveness of combined multi-target siRNA anticancer approach and described new highly effective serum stable nanomaterial vehicle for gene-based drugs.
Highlights
The discovery of the double-stranded RNA-mediated interference (RNAi), which is a simple and rapid post-transcriptional method of inhibition of gene expression in organisms, represents an important tool in medicine
Using siRNAs as anticancer agents requires intravenous administration associated with several intrinsic problems, such as their degradation by plasma nucleases, poor ability to penetrate into cells and rapid systemic removal by glomerular filtration
The cancer cell growth inhibition caused by dendrimers, siRNAs, 5-FU and their combinations was assessed using MTT cytotoxicity assay based on Mossman et al [31] with 3-[4,5-2-yl]-2-5-diphenyltetrazolium bromide (Sigma-Aldrich, Poznan, Poland)
Summary
The discovery of the double-stranded RNA-mediated interference (RNAi), which is a simple and rapid post-transcriptional method of inhibition of gene expression in organisms, represents an important tool in medicine. Dendrimers, a class of nanoscale hyperbranched polymers with radial symmetry, represent one of the most promising tools for delivering of therapeutic genetic material into cancer cells [4,5,6,7] Their well-defined structure with multivalent surface groups being, for instance, positively charged allows siRNAs to bind to them, thereby forming stable complexes called dendriplexes. Two major studies concerning binding properties of CPD towards siRNAs involved diethylamine-terminated dendrimers and were focused on siRNA directed against BCR [19] and anti-apoptotic genes (MCL-1, BCL-xL and BCL-2) [20] Both studies report stable complex formation between CPD and siRNAs, with complexes being positively charged and secondary structure of siRNA unaltered. AE2G4H+. 31P{1H} NMR (CD3OD): δ 9.0 (P0), 62.6 (br s P1, P2, P3), 69.5 (P4)
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