Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serumadsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-coglycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.
Gene transfer has many potential applications in basic and applied sciences. In vitro, DNA delivery can be enhanced by increasing the concentration of DNA in the cellular microenvironment through immobilization of DNA to a substrate that supports cell adhesion. Substrate-mediated delivery describes the immobilization of DNA, complexed with cationic lipids or polymers, to a biomaterial or substrate. As surface properties are critical to the efficiency of the surface delivery approach, selfassembled monolayers (SAMs) of alkanethiols on gold were used to correlate surface chemistry of the substrate to binding, release, and transfection of non-specifically immobilized complexes. Surface hydrophobicity and ionization were found to mediate both DNA complex immobilization and transfection, but had no effect on complex release. Additionally, SAMs were used in conjunction with soft lithographic techniques to imprint substrates with specific patterns, resulting in patterned DNA complex deposition and transfection, with transfection efficiencies in the patterns nearing 40%. Controlling the interactions between complexes and substrates, with the potential for patterned delivery, can be used to locally enhance or regulate gene transfer, with applications to tissue engineering scaffolds and transfected cell arrays.
Novel pH-sensitive gel-forming pentablock copolymers based on commercially available Pluronic (poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), PEO-b-PPO-b-PEO) triblock copolymers and cationic diblock copolymers based on polyethylene glycol) methyl ether (PEGME) were synthesized by oxyanionic polymerization. Polymerization of the cationic moiety, poly((diethylamino)-ethyl methacrylate), PDEAEM, was initiated by a difunctional potassium alcoholate of the triblock Pluronic copolymer F127 (PEO106-PPO69-PEO106) or PEGME. The difunctionality of the initiation using the triblock macroinitiator, indicating formation of a pentablock copolymer rather than a tetrablock copolymer, was verified by functionalized termination of the living polymer chains. Critical micellization temperatures (cmt) of the synthesized polymers were obtained from differential scanning calorimetry for the pentablock materials. The pentablock copolymers retained the thermoreversible gel-forming properties of Pluronic F127 as well as similar cmt values. The polydispersity of both the diblock and pentablock copolymers was similar to the macroinitiators, indicating a very low polydispersity associated with the addition of the cationic PDEAEM blocks. Both of the materials show pH-sensitive release behavior, whereas the native polymers do not. ABSTRACT: Novel pH-sensitive gel-forming pentablock copolymers based on commercially available Pluronic (poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), PEO-b-PPO-b-PEO) triblock copolymers and cationic diblock copolymers based on poly(ethylene glycol) methyl ether (PEGME) were synthesized by oxyanionic polymerization. Polymerization of the cationic moiety, poly((diethylamino)-ethyl methacrylate), PDEAEM, was initiated by a difunctional potassium alcoholate of the triblock Pluronic copolymer F127 (PEO 106-PPO69-PEO106) or PEGME. The difunctionality of the initiation using the triblock macroinitiator, indicating formation of a pentablock copolymer rather than a tetrablock copolymer, was verified by functionalized termination of the living polymer chains. Critical micellization temperatures (cmt) of the synthesized polymers were obtained from differential scanning calorimetry for the pentablock materials. The pentablock copolymers retained the thermoreversible gel-forming properties of Pluronic F127 as well as similar cmt values. The polydispersity of both the diblock and pentablock copolymers was similar to the macroinitiators, indicating a very low polydispersity associated with the addition of the cationic PDEAEM blocks. Both of the materials show pH-sensitive release behavior, whereas the native polymers do not.
Direct muscular injection of IGF-I effectively increases EOM force generation without the potential biomechanical hazards of surgery such as permanently altered muscle length or insertional position on the globe.
All modern web browsers -Internet Explorer, Firefox, Chrome, Opera, and Safari -have a core rendering engine written in C ++ . This language choice was made because it affords the systems programmer complete control of the underlying hardware features and memory in use, and it provides a transparent compilation model. Unfortunately, this language is complex (especially to new contributors!), challenging to write correct parallel code in, and highly susceptible to memory safety issues that potentially lead to security holes.Servo is a project started at Mozilla Research to build a new web browser engine that preserves the capabilities of these other browser engines but also both takes advantage of the recent trends in parallel hardware and is more memory-safe. We use a new language, Rust, that provides us a similar level of control of the underlying system to C ++ but which statically prevents many memory safety issues and provides direct support for parallelism and concurrency.In this paper, we show how a language with an advanced type system can address many of the most common security issues and software engineering challenges in other browser engines, while still producing code that has the same performance and memory profile. This language is also quite accessible to new open source contributors and employees, even those without a background in C ++ or systems programming. We also outline several pitfalls encountered along the way and describe some potential areas for future improvement.
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