Abstract
Local gene delivery systems utilizing RNA interference technology are a promising approach for therapeutic applications where site-specific release of agents is desired. Polyelectrolyte multilayers (PEMs) can be constructed using the layer-by-layer (LbL) technique and serve as a depot for bioactive substances, which can then be released in a controlled manner. Multilayers of hyaluronic acid/poly(ethylenimine) HA/PEI were built up with different numbers of bilayers and PEI-siRNA particles were embedded in bioactive layers for gene silencing. The increase of the bilayers and the release of siRNA particles were demonstrated by fluorescence intensity measurement with a fluorescence reader. Two different LbL techniques were tested for the reduction of ICAM–1 expression in EA.hy926: PEM build-up by dipping or drying steps, respectively. Herein, the drying technique of the bioactive layers with ICAM siRNA mediated a significant reduction of the ICAM–1 expression from 3 to 24 bilayers. The fluorescent siRNA release study and the re-culturing of the HA/PEI films demonstrated a release of the transfection particles within the first hour. The advantage of dried built-up PEMs compared to a dried monolayer of PEI-siRNA particles with the same siRNA concentration was a significant higher amount of viable cells.
Highlights
The RNA interference (RNAi) mechanism is a powerful and specific technique in molecular biology and shows high potential for therapeutic applications, including cardiovascular diseases (CVD), infectious diseases and cancer [1,2,3,4,5,6,7,8]
PEI)2(HA/PEI-short interfering RNA (siRNA))3,10 coating showed a minimal decrease of Intercellular Adhesion Molecule 1 (ICAM–1) expression with remaining 89% and 86% but was not significant compared to the control (Fig 2)
A transfection efficiency of 21% was measured with Polyelectrolyte multilayers (PEMs) PEI(HA/PEI)2(HA/PEI-siRNA AF 488 (siRNAf))3 and 50% with PEM PEI(HA/PEI)2(HA/PEI-siRNAf)10 (Fig 3)
Summary
The RNA interference (RNAi) mechanism is a powerful and specific technique in molecular biology and shows high potential for therapeutic applications, including cardiovascular diseases (CVD), infectious diseases and cancer [1,2,3,4,5,6,7,8]. RNAi was first discovered by Fire et al in 1998 in C. elegans with double-stranded RNA causing the silence of complementary messenger RNA sequence [9]. This self-defense mechanism in Eukarya is known for preventing infections and following gene integrity by pathogens and regulating gene expression processes. The ability to artificially produce short interfering RNA (siRNA) with 21–23 nucleotides which binds specific to the target mRNA is a promising approach for various therapeutic applications with the aim of transient gene silencing [10]. The delivery of siRNA to the target tissue or cells is one of the major challenges.
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