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.

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.

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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.

Non-radioactive tracer tagged proppant technology has been used successfully in over 200 vertical wells to obtain fracture height and gravel pack coverage, but had yet to be modeled or tested in horizontal wells, where the borehole and fracture geometry is radically different. The purpose of this paper is to demonstrate the effectiveness of this new technology to locate proppant placement and to evaluate the efficiency of perforations and clustering in fracturing operations in horizontal wells. The new technology utilizes a pulsed neutron capture (PNC) logging tool to detect the tagged proppant, which has a high thermal neutron capture cross-section. Monte Carlo software is utilized to simulate the responses of different pulsed neutron measurements to vertical fracture planes along a horizontal wellbore. The tool responses were analyzed and the best PNC measurement parameters were determined for locating proppant placement in the fractures, and hence for determining perforation and cluster efficiency. A field test was the conducted to validate modeling results in a horizontal well with multi-stage fracturing operations. Modeling data and the field log example show that it is feasible to use the taggant and a PNC tool to locate proppant placement and determine the efficiency of perforations and clustering in horizontal wells. Furthermore, the analysis of the depth intervals of tracer signals and the known axial and radial resolutions of the PNC measurements show that the depth interval of tracer signals along the wellbore may provide a qualitative indicator of one or more of the following: (1) the presence of multiple fractures opposite perforation(s), (2) the angle between the wellbore axis and the fracture plane for ideal conditions (e.g. the more narrow the tracer signal along the log, the closer the fracture plane is to being perpendicular to the wellbore axis), and/or (3) proppant placement in the borehole region. Moreover, a new near-wellbore connectivity index has been developed based on identifying and using the most suitable PNC log parameters for the evaluation. The index indicates how well the induced fracture is connected to the wellbore at locations of all perforations/clusters. The field test demonstrated that this new technology met all of the operator's objectives, with tracer signals clearly observed in all stages. This paper will present the modeling work and the results from the field test. The new non-radioactive proppant tracer technology provides for the first time the ability to evaluate proppant location in horizontal wells without encountering the health, safety, and environmental issues associated with using radioactive tracers. The determination of proppant placement and perforation efficiency, and a near-wellbore connectivity index, can be employed to evaluate the success and effectiveness of individual perforations and stages, and to optimize future completion designs and processes for enhancing hydrocarbon recovery.

Cement quality is typically determined through the use of sonic logging tools, more commonly known as cement bond logs (CBLs), or more recently ultrasonic imaging tools (USITs). In general, these tools have served the industry well over time, but with the advent of new and exotic cement blends, as well as multistage cement jobs in today's unconventional horizontal wells, the quality and even location of the cement has become more problematic for basic CBL/USIT tools to detect. In addition, these tools are ineffective through multiple uncemented casing strings. A novel method to detect cement was developed as an offshoot of a technology used for detecting proppant in hydraulically fractured wells. This technique uses a non-radioactive tracer which exhibits a high thermal neutron capture cross section that is then incorporated into the proppant grains during manufacture. The proppant can then be detected using standard neutron-logging tools, at any time during the well's life. By incorporating small volumes of this detectible proppant into the cement slurry, the cement can then be detected using the same logging tools. This leads to identification of the top of cement, as well as the cement quality. If desired, the taggant can be staged such that the top and bottom of a cement stage can be detected. This paper will first review the industry concerns with cement detection. It will then discuss the principles and theory behind how the taggant works, both for basic proppant detection, as well as the novel application as a vehicle for cement detection. This will also include lab testing showing no impact of the tagged proppant on cement performance. The authors will conclude by presenting several case histories of cement detection, including two horizontal well applications, one each in the Permian and Canada. A third case history will also be presented in which the cement was detected through multiple strings of uncemented casing, to verify success of a cement squeeze in a surface casing remediation. This new technique allows for cement detection in wells in which conventional CBL/USITs are difficult to interpret, including detection of exotic cement blends, and through multiple strings of casing. This allows for more confidence in cement isolation, particularly in today's unconventional wells, where isolation of uphole formations is critical. This paper will be useful for drilling and completion engineers who are concerned with their ability to confirm cement quality, as well as production engineers who must perform remedial cementing operations.

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