Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1 %, and 8.1 %, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration.
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Hydrogels have been widely investigated for localized, sustained gene delivery because of the similarity of their physical properties to native extracellular matrix and their ability to be formed under mild conditions amenable to the incorporation of bioactive molecules. The objective of this study was to develop bioactive hydrogels composed of macromolecules capable of enhancing the efficiency of non-viral vectors. Hybrid hydrogels were prepared by simultaneous enzymatic and Michael-type addition crosslinking of reduced fibrinogen and an acrylated amphiphilic block copolymer, Tetronic T904, in the presence of dithiothreitol (DTT) and thrombin. T904/fibrin hydrogels degraded by surface erosion in the presence of plasmin and provided sustained release of polyplex vectors up to an order of magnitude longer than pure fibrin gel control. In addition, the rate of gel degradation and time-course of polyplex vector release were readily controlled by varying the T904/fibrinogen ratio in the gel composition. When added to transfected neuroblastoma (N2A) cells, both native T904 itself and hydrogel degradation products significantly increased polyplex transfection efficiency with minimal effect on cell viability. To evaluate gel-based transfection, N2A cells encapsulated in small fibrin clusters were covered by or suspended within polyplex-loaded hydrogels. Cells progressively degraded and invaded the hybrid hydrogels, exhibiting increasing gene expression over 2 weeks and then diminishing but persistent gene expression for over 1 month. In conclusion, these results demonstrate that T904/fibrin hybrid hydrogels can be promising tissue engineering scaffolds that provide local, controlled release of non-viral vectors in combination with the generation of bioactive gel degradation products that actively enhance vector efficiency. Copyright © 2014 John Wiley & Sons, Ltd.
Summary More than 100 billion lbm of proppant are placed annually in wells across the globe, with the majority in unconventional reservoirs. The location of the proppant in these horizontal wells and formations is critical to understanding reservoir drainage, well spacing, and stage spacing. However, for many years proppant detection has primarily been limited to near-wellbore measurements. A novel method to detect proppant in the far field has been developed and is the subject of this paper. The proppant-detection method developed uses electromagnetic (EM) methods. This technology entails using a transmitter source and an array of electric- and magnetic-field sensors at the surface. A current signal with a unique wave form and frequency is transmitted to the bottom of the wellbore via a standard electric-line (E-line) unit. In addition, an electrically conductive proppant is pumped into the stage(s) of interest. The electric and magnetic fields are measured both before and after the detectable proppant stages, and a novel analysis method is then used to process and invert these differenced data to create an image of the propped reservoir volume (PRV). This technology is the product of years of development of computer models capable of forward modeling this technique. Once this modeling was completed, an initial field test was performed in west Texas (WTX), with a preliminary analysis of this work presented in a previous paper (Palisch et al. 2016). Since that paper, however, additional processing of the data has yielded a much-more-detailed image of the proppant location in this Bone Springs well. In addition, a subsequent field application has been performed in a major basin in the northeastern US. Multiple stages received detectable proppant of varying stage volumes, and the analysis has also shown a detailed image of the proppant location in that wellbore. Furthermore, the initial field test in WTX used only electric-field sensors, whereas this latest test used both electric- and magnetic-field receivers. The authors’ numerical simulations coupled with the field results indicate the percentage difference between prefracture and post-fracture results is two times higher using magnetic- vs. electric-field sensors. This paper will review the technology development and methods, will present the latest imaging from the initial WTX test, and will describe the latest learnings from the most-recent field test. This paper should be beneficial to all completions and development personnel who are interested in knowing where proppant is in their fractures. This technology has the potential to assist in understanding well drainage and spacing, stage and perforation-cluster spacing, vertical fracture coverage, and the effect of fracture-design changes.
Background Amphiphilic block copolymers acting as biological response modifiers provide an attractive approach to improving the transfection efficiency of polycationic polymer/DNA complexes (polyplexes) by altering cellular processes crucial to efficient transgene expression. Methods The objective of this study was to investigate the effect of the poloxamine Tetronic T904, a 4-arm polyethylene oxide / polypropylene oxide block copolymer, on polyplex transfection and determine its mechanism of action by analyzing cellular uptake of polyplex, nuclear localization of plasmid, and RNA transcript production. Results T904 significantly increased the transfection efficiency of polyplexes based on 25 kDa branched polyethylenimine in a dose-dependent manner in the presence of serum in C6 glioma cells, as well as human fibroblasts and mesenchymal stem cells. The activity of T904 was not promoter-dependent, increasing expression of reporter genes under both CMV and SV40 promoters. While T904 did not affect internalization or nuclear uptake of plasmid, mRNA expression levels from both promoters showed dose-dependent increases that closely paralleled increases in gene expression. Conclusions This study demonstrates that T904 significantly increases polyplex transfection efficiency and suggests a mechanism of increased transcriptional activity. As a 4-arm, hydroxyl-terminated polymer, T904 is amenable to a variety of end group functionalization and covalent crosslinking strategies that have been developed for preparing hydrogels from multi-arm polyethylene glycol, making it particularly attractive for scaffold-mediated gene delivery.
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