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.