Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells.

Polyethyleneimine (PEI) has been used extensively for transient gene expression (TGE) in mammalian cell cultures. However, the relationship between DNA/PEI complex preparation and their biological activity has not been fully established. Here, a systematic study of DNA/PEI complexes, their physicochemical properties during formation and their transfection efficiency was performed on a virus-like particle (VLP) production platform. The same chemically defined cell culture medium for DNA/PEI complex formation was used as an alternative to simple ionic solutions to minimize changes in complex properties during transfection. Upon formation, an initial concentration of 1E + 10 DNA/PEI complexes/mL underwent partial aggregation with an average size of 300 nm. The participation of NaCl ions in the evolution of complexes was analyzed by X-ray spectroscopy, stressing the relevance of complexing media composition in TGE strategies. After 15 min incubation, 250 complexes plus aggregates per cell were estimated at the time of transfection. Such heterogeneous preparations cannot be easily characterized; subsequently, nanoparticle tracking analysis (NTA) and cryo-electron microscopy were combined to achieve a complete picture of the preparation. Finally, the contribution of each DNA/PEI complex subpopulation was tested by drug inhibition endocytosis. Interestingly, all complexes delivered DNA efficiently and high size aggregates, which enter through macropinocytosis, when inhibited presented a major contribution to transfection efficiency. There is a need to understand the physicochemical factors that participate in DNA delivery protocols. Hence, this study provides new insights into the characterization of DNA/PEI complexes that will assist in more productive and reproducible TGE strategies.

Lipid-based nanovesicles for nanomedicine.

Molecular self-assembly has enabled the fabrication of biologically inspired, advanced nanostructures as lipid-based nanovesicles (L-NVs). The oldest L-NVs, liposomes, have been widely proposed as potential candidates for drug delivery, diagnostic and/or theranostic applications and some liposome-based drug products have already stepped from the lab-bench to the market. This success is attributed to their ability to encapsulate both hydrophobic and/or hydrophilic molecules, efficiently carry and protect them within the body and finally deliver them at the target site. These positive features are also coupled with high biocompatibility. However, liposomes still present some unsolved drawbacks, such as poor colloidal stability, short shelf-life, restricted and expensive conditions of preparation because of the inherent nature of their fundamental constituents (phospholipids). The new tools available in the self-assembly of controlled molecules have significantly advanced the field of L-NV design and synthesis, and non-liposomal L-NVs have been recently developed; this new generation of nanovesicles can represent a paradigm shift in nanomedicine: they may complement liposomes, showing their advantages and overcoming most of their drawbacks. Clearly, being still young, their rocky way to the clinic first and then to the market has just started and it is still long, but they have all the potentialities to reach their objective target. The purpose of this review is to first present the large plethora of L-NVs available, focusing on this new generation of non-liposomal L-NVs and showing their similarities and differences with respect to their ancestors (liposomes). Since the overspread of a nanomaterial to the market is also strongly dependent on the availability of technological-scale preparation methods, we will also extensively review the current approaches exploited for L-NV production. The most cutting-edge approaches based on compressed fluid (CF) technologies will be highlighted here since they show the potential to represent a game-change in the production of L-NVs, favouring their step from the bench to the market. Finally, we will briefly discuss L-NV applications in nanomedicine, looking also for their future perspectives.

Endoscopic treatment of postoperative fistulas resistant to conservative management using biological fibrin glue.

BACKGROUND AND STUDY AIMS: Postoperative fistulae occur frequently in standard surgical practice, but there is no general agreement on how to treat them. We summarize here our experience with endoscopic treatment. PATIENTS AND METHODS: Postoperative digestive fistulae resistant to conservative treatment, in 15 patients, are retrospectively reviewed. Our series included two internal fistulas: (one rectovesical, and one high-output pleuroesophagic), and 13 external fistulas (one low-output gastrocutaneous, two low-output esophagocutaneous, seven low-output enterocutaneous, and three high-output enterocutaneous). After failure of conservative treatment, the fistulas were endoscopically located and 2 - 4 ml of reconstituted fibrin glue, Tissucol 2.0 at 37 degrees C, was injected through a catheter. RESULTS: The mean age of the patients was 61.2 years (38 - 86), and 60 % were men. Of the fistulas, 26.6 % were of the high-output type. The mean healing time was 16 days (5 - 40), and a mean of 2.5 sessions per patient were required (1 - 5). Complete sealing of fistulas was achieved in 86.6 % of cases; (87.5 % of the low-output and internal fistulas, and 55 % of the high-output fistulas). After follow-up ranging between 2 months and more than 3 years, only one of the sealed fistulas reopened. No complications were encountered. Overall mortality was 13.3 % (two out of 15), but in only one patient was this related to clinical deterioration because of the persistence of the fistula. CONCLUSIONS: We think that conservative treatment should not be prolonged beyond 14 days and that endoscopic treatment should be performed at that stage. Endoscopic sealing treatment achieves a very high success rate, without complications and at a lower cost. It could probably reduce the hospital stay, and avoid some unnecessary surgical interventions. Appropriate multicenter randomized trials are needed to confirm these results.

Stereoisomerism of molecular multipropellers. 1. Static stereochemistry of bis- and tris-triaryl systems.

The static stereoisomerism of bis- and tris-triaryl systems has been analyzed by a systematic stereochemical analysis, and the resulting theoretical predictions have been experimentally confirmed by using reversed-phase HPLC and ESR and (1)H NMR spectroscopies with a family of seven distinct polychlorinated aromatic multipropellers. To analyze the static stereochemistry of these molecules, we have developed a specific procedure that uses a symmetry-adapted symbolic notation, allowing the theoretical prediction of both the number and symmetry of the isomers of the investigated molecules. Due to the steric hindrance introduced by the presence of bulky chlorine substituents, (all) conformational isomers can be characterized experimentally by several independent techniques confirming the theoretical stereochemical predictions. The different propeller moieties that constitute the molecule appear to be nearly independent of each other. Consequently, most of the observed isomers show comparable populations in solution at room temperature.

Stereoisomerism of molecular multipropellers. 2. Dynamic stereochemistry of bis- and tris-triaryl systems.

The dynamic stereochemistry of bis- and tris-triaryl systems, the most simple "molecular multipropellers", is discussed on the basis of an extension of a systematic stereochemical analysis based on a symmetry-adapted symbolic notation developed specifically for these molecules. A suitable theoretical basis for our study is provided by the classical hypotheses concerning the dynamics of simple triaryl systems as formulated by Mislow and co-workers (J. Am. Chem. Soc. 1973, 95, 1535-1547), which, once applied to molecular multipropellers, show the existence of two modes of rearrangement for each propeller. Interconversion graphs for all molecules under study, covering a wide span of structural complexity, are presented. A complete NMR study of a two- and a three-propeller molecule indicates that all experimentally observable exchange pathways are indeed predicted by theoretical analysis. Moreover, quantitative analysis of 2D-EXSY experiments affords the activation energy of the subset of pathways that give rise to observable interconversions on the NMR time scale. Assuming that two-ring flips are the threshold mechanism for individual propeller interconversion, the experimental evidence indicates a preference for the flip of the central ring and one of the outer rings over the flip of two outer rings.

[Polyps of the biliary tract: is their preoperative diagnosis possible?]. / Pólipos de la vía biliar: es posible su diagnóstico preoperatorio?

Bile duct polyps are a very uncommon cause of obstructive jaundice. We present our experience of three patients diagnosed in the last 10 years. Initial presentation usually takes the form of obstructive jaundice associated with abdominal pain, which simulates biliary lithiasis. The diagnosis is usually surgical. Although in some cases radiological studies and endoscopic retrograde cholangiopancreatography (ERCP) may sometimes detect bile duct polyps, exact diagnosis before surgery is very unusual. The radiological signs that suggest the existence of a bile duct polyp in the ERCP seem to be the presence of repletion defects, fixed unilaterally to the biliary conduit, without meniscus and without circumferential stenosis of the affected conduit. The most frequently found polyps are fibroinflammatory, and less frequently adenomatous.