PF6 (100 µM stock solution) was mixed with siRNA (10 µM stock solution) in MQ water in one-tenth of final treatment volume (i.e. 50 µl), using MR30 in serum free media or MR40 in serum experiments. Complexes were formed for 30 min at RT and added to cells, grown to 60% confluence in 24-well plate, in 450 µl growth media. After 4 h, 1 ml of fresh media was added to wells and cells were incubated for indicated times. Treatments with LF2000 and RNAiMAX were conducted in accordance with recommendations from manufacturer (Invitrogen), using 1 µl/well for 50 nM siRNA in 24-well plates. TransductinTM was used according to guidelines (IDT Technologies), using 6–7 µM reagent with 100 nM siRNA but performing experiments in serum supplemented or serum free media (instead of Q-serum devoid of GAGs). In luciferase experiments, cells were lysed using 100 µl of 0.1% Triton X-100 in Hepes Krebbs Ringer buffer. After 30 min lysis on ice, luciferase expression was measured using Promega Luciferase Kit on 96-well Glomax luminometer (Promega). If using Cy5-labled siRNAs, lysates were first analyzed by fluorometry using a Spectra Max Gemini (Molecular Devices) prior to luciferase measurements. For in vivo treatment trials, PF6/siRNA particles (MR30) were formed in MQ-water in half of the injection volume (i.e. 100 µl), using 2 mM PF6 and 0.5 mM siRNA stock solutions. After 30 min incubation, 100 µl of 10% glucose or 10.8% mannitol solution was added to the particles. Two hundred micro liter solution was injected into the tail-vein of mice. Hydrodynamic infusions in tail vein of mice were carried out using 1 mg/kg siRNA in 2 ml saline buffer injected with high pressure. Hydrodynamic mean diameter of PF6/siRNA particles was determined (using a refractive index of 1.338) by DLS studies (Zetasizer Nano ZS apparatus, Malvern Instruments) and z-potential was measured using the same instrument.