Role of temperature-independent lipoplex-cell membrane interactions in the efficiency boost of multicomponent lipoplexes

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4?°C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.     

Evaluation of the MDR1, ABCG2, Topoisomerases IIalpha and GSTpi gene expression in patients affected by aggressive mantle cell lymphoma treated by the R-Hyper-CVAD regimen

The genomic profile of mantle cell lymphoma (MCL) has been reported to be significantly different from that of other indolent lymphoproliferative disorders, Topoisomerase IIalpha, glutathione-s-transferasepi (GSTpi) and ABCG2 (BCRP) chemoresistance genes being over-expressed in MCL. In our study, expression levels of the above mentioned genes plus MDR1 were tested on bone marrow samples from 20 patients treated with Rituximab plus hyper-CVAD regimen, in order to evaluate a possible impact of the chemoresistance phenomenon on this promising treatment regimen. All patients expressed ABCG2 and MDR1 genes; 85% of cases expressed GSTpi and topoisomerase IIalpha. Only ABCG2 were over-expressed in comparison both with marrow from healthy donors and tonsilar CD5+/CD20+ lymphocytes (adopted as normal counterpart of the neoplastic population). The overall response rate of the entire series was 87.5%, with 44% of complete responses. Fifty-seven percent of patients achieved the clearance of minimal residual disease. Levels of tested genes did not condition either quality of clinical response or PFS (76% at 24 months). Nevertheless, an ABCG2 higher expression appeared associated with a worse PFS and levels of this gene paralleled the status of minimal residual disease. A further evaluation of ABCG2 expression in larger series of MCL patients would be suitable.     

Chronic phencyclidine administration reduces the expression and editing of specific glutamate receptors in rat prefrontal cortex

Phencyclidine (PCP) induces a form of psychosis that mimics naturally occurring schizophrenia in the most relevant domains of the psychopathology. In this report, we investigated the effect of chronic treatment with PCP on expression and RNA editing of alpha-amino-propionic acid (AMPA) and kainate (KA) glutamate receptor (GluR), in the rat prefrontal cortex and the hippocampus. We found that chronic, but not acute, PCP treatment decreased GluRs expression in the rat prefrontal cortex but not in the hippocampus. In particular, the mRNA coding for GluR2 and GluR3 subunits were reduced by 50%, whereas those coding for KA GluR5 and GluR6 were decreased by 30%. In addition, we observed a decrease of the editing levels of the R/G site in the flop form of both GluR2 and GluR3 and a significant increase in the editing level of GluR6 Q/R site. The variation in the editing level of the R/G sites suggests that chronic PCP treatment induced the formation of glutamate receptor subunits with slower resensitization kinetics and, with respect to kainate receptors, an increase in the Q/R editing level might generate receptor channels with a lower permeability to cations. Combining all the data, it can be inferred that the PCP treatment induced a specific and site-selective reduction of glutamatergic neurotransmission in the prefrontal cortex but not in the hippocampus.