Poly(D,L-lactide-co-glycolide acid) nanoparticles for DNA delivery: waiving preparation complexity and increasing efficiency

When designing a nonviral gene delivery system based on polymeric nanoparticles (NPs), it is important to keep in mind obstacles associated with future clinical applications. Simplifying the procedure of NPs production and taking toxicity into account are the most important issues that need to be addressed. Toxicity concerns in clinical trials may be raised when using additives such as cationic polymers/lipids, buffering reagents, and proteins. Therefore, the aim of this study was to simplify the formulation of poly (lactide-co-glycolide) acid NPs by shortening steps such as sonication time and by avoiding the use of additives while preserving its efficiency. NPs (300 nm) were formulated using a modified w/o/w technique with DNA entrapment efficiency of 80%. Once achieving such NPs, formulation parameters such as DNA loading, release kinetics, DNA integrity and bioactivity, uptake by cells, and toxicity were addressed. The NPs were readily taken by several cell lines and were localized mostly in their endo-lysosomal compartments. The NPs did not affect cells viability. Most importantly, transfection studies in COS-7 and Cf2th cells resulted with a 250-fold protein expression levels when compared with the control. These expression levels are higher than ones achieved with more complicated NPs systems, demonstrating the efficiency of our simplified NPs for gene delivery.     

VEGF162, a new heparin-binding vascular endothelial growth factor splice form that is expressed in transformed human cells

The splice forms of vascular endothelial growth factor (VEGF) differ in biological properties such as the receptor types that they recognize and their interaction with heparan sulfate proteoglycans. We have identified a new VEGF mRNA splice form encoding a VEGF species containing 162 amino acids (VEGF(162)) in human A431 ovarian carcinoma cells. This novel mRNA contains the peptides encoded by exons 1-5, 6A, 6B, and 8 of the VEGF gene. Recombinant VEGF(162) is biologically active. It induces proliferation of endothelial cells in vitro and angiogenesis in vivo as determined by the alginate bead assay. VEGF(162) binds less efficiently than VEGF(145) but more efficiently than VEGF(165) to a natural basement membrane produced by corneal endothelial cells. VEGF(138), an artificial VEGF form that contains exon 6B but lacks exons 6A and 7, did not bind to this basement membrane at all, indicating that exon 6B probably interferes with the interaction of exon 6A with heparin and heparan sulfate proteoglycans.     

Alginate-chitosan complex coacervation for cell encapsulation: effect on mechanical properties and on long-term viability

The use of chitosan in complexation with alginate appears to be a promising strategy for cell microencapsulation, due to the biocompatibility of both polymers and the high mechanical properties attributed by the use of chitosan. The present work focuses on the optimization and characterization of the alginate-chitosan system to achieve long-term cell encapsulation. Microcapsules were prepared from four types of chitosan using one- and two-stage encapsulation procedures. The effect of reaction time and pH on long-term cell viability and mechanical properties of the microcapsules was evaluated. Using the single-stage encapsulation procedure led to increase of at least fourfold in viability compared with the two-stage procedure. Among the four types of chitosan, the use of high molecular weight (MW) chitosan glutamate and low MW chitosan chloride provided high viability levels as well as good mechanical properties, i.e., more than 93% intact capsules. The high viability levels were found to be independent of the reaction conditions when using high MW chitosan. However, when using low MW chitosan, better viability levels (195%) were obtained when using a pH of 6 and a reaction time of 30 min. An alginate-chitosan cell encapsulation system was devised to achieve high cell viability levels as well as to improve mechanical properties, thus holding great potential for future clinical application.     

CIRCADIAN PATTERN OF SIMULATED FLIGHT PERFORMANCE OF PILOTS IS DERIVED FROM ULTRADIAN COMPONENTS *

Studies suggest some physiologic, cognitive, and behavioral 24h rhythms are generated by cyclic components that are shorter in period than circadian. The aim of this study was [1] to examine the hypothesis that 24h human performance rhythms arise from the integration of high-frequency endogenous components and [2] to quantify the contribution of each higher frequency component to the phenotype of the rhythm. We monitored the performance of 9 experienced pilots by employing an array of cognitive-based tests conducted in a flight simulator so that, over the 6-day experiment, data were obtained for each 2h interval of the 24h. The activity-rest schedule of the subjects, no matter the exact clock time schedule of sleep and activity, always consisted of 14h activity (when they carried out regular professional duties) and 10h rest, with at least 8h of sleep. The simulated combat scenarios consisted of simple and complex tasks associated with target interception, aircraft maneuvering, and target shooting and downing. The results yielded two indices: the number of prominent periodicities in the time series and the relative magnitude of the amplitude of each relative to the construction of the composite 24h waveform. Three cyclic components (8h, 12h, and 24h) composed the observed 24h performance pattern. The dominant period and acrophase (peak time) of the compound output rhythm were determined by the interplay between the amplitudes of the various individual ultradian components. Task complexity (workload) increases the expression of the ultradian entities in the 24h pattern. We constructed a model composed of the multiple ultradian components; the composite output defined a “time span” (of 2h-4h duration) as opposed to an exact “time point” of high and low performance, endowing elevated functional capability. (Chronobiology International, 18(6), 987-1003, 2001)     

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs,an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.     

Continuous release of endostatin from microencapsulated engineered cells for tumor therapy

Research studies suggest that tumor-related angiogenesis contributes to the phenotype of malignant gliomas. We assessed the effect of local delivery of the angiogenesis inhibitor endostatin on human glioma cell line (U-87MG) xenografts. Baby hamster kidney (BHK) cells were stably transfected with a human endostatin (hES) expression vector and were encapsulated in alginate-poly L-lysine (PLL) microcapsules for long-term delivery of hES. The release of biologically active endostatin was confirmed using assays of bovine capillary endothelial (BCE) proliferation and of tube formation. Human endostatin released from the microcapsules brought about a 67. 2% inhibition of BCE proliferation. Furthermore, secreted hES was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial cells stably transfected with KDR, a tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts' weight 21 days post treatment. This inhibition was achieved by only 150.8 ng/ml human endostatin secreted from 2 x 10(5) encapsulated cells. Encapsulated endostatin-secreting cells are effective for the treatment of human glioblastoma xenografts. Continuous local delivery of endostatin may offer an effective therapeutic approach to the treatment of a variety of tumor types.