Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells

Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement – a stage between initial cellular internalization and final gene silencing of siRNA delivery systems

The Hemobag: the modern ultrafiltration system for patients undergoing cardiopulmonary by pass.

Abstract Background The return of extracorporeal circuit blood at the termination of cardiopulmonary bypass (CPB) is an important feature of blood conservation during cardiac surgery procedures globally. We report our initial clinical evaluation of the Hemobag system a blood-salvaging device designed for whole blood recovery of residual post-CPB volume. Methods Residual whole blood is hemoconcetrated through the multipass “recovery loop” circuit separate from the CPB and collected in the Hemobag System. This allows the surgeons to continue with surgery, decannulate, and administer protamine simultaneously while the Hemobag is in use and the CPB circuit remains safely primed. We have compared 25 patients receiving the Hemobag to a control group of 25 patients treated with the cell washer that represented our previous standard of care method of circuit blood-salvaging technique. Results The Hemobag system provided significantly higher hemoglobin, hematocrit, fibrinogen, albumin, and total protein levels in the final product reducing the amount of wasted autologous blood cells. There were no device-related complications. There were no significant differences in terms of blood utilization, chest tube drainage and clinical outcomes over the entire postoperative period among groups. Conclusions These results suggest that the Hemobag system is a safe and efficient method to multipass hemoconcentrate the residual diluted blood of the CPB circuit. The Hemobag has demonstrated its ability to maximize the composition of the residual CPB volume to achieve the best possible post-CPB hemoglobin, plasma protein and coagulation factors profile for the patient respect to CW.</p

Aortic valve replacement with minimal extracorporeal circulation versus standard cardiopulmonary bypass.

The purpose of this study was to evaluate the safety and the clinical outcomes of aortic valve replacement (AVR) performed with minimally invasive extracorporeal circulation (miniECC) technique vs. standard cardiopulmonary bypass (CPB). From February 2006 to December 2007 a total of 181 isolated AVR were performed, of these 53 patients were operated using minimal extracorporeal circulation system and 128 patients were operated using the standard CPB. Demographic characteristics and operative data were similar in both groups except for EuroSCORE (P1.7 m(2) and who underwent traditional CPB in respect to miniECC system. Postoperatively renal injury, atrial fibrillation episodes, neurologic event rate, ICU and hospital stay length were similar in both groups. The miniECC is suitable for AVR providing good clinical results but the present results should not identify the miniECC system to be superior to the conventional CPB