Efficacy and mechanism of poloxamine‐assisted polyplex transfection

Abstract

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.

Cationic, amphiphilic copolymer micelles as nucleic acid carriers for enhanced transfection in rat spinal cord

“…In our own previous work, we have found that a related Tetronic block copolymer of similar HLB to PgP can increase gene expression from plasmid vectors by as much as an order of magnitude by increasing steady-state transgene mRNA levels. [46]. To further characterize the effect of HLB on transfection efficiency and gain further insight into its mechanistic basis, we synthesized PgP using different molecular weights of PLGA (25kDA and 50 kDa) in ongoing studies.…”

Poloxamine/fibrin hybrid hydrogels for non-viral gene delivery

“…When added to transfected cells, both native T904 and gel degradation products significantly increased polyplex transfection efficiency with minimal effects on cell viability. This outcome, previously reported for both native Pluronics and T904 (Sriadibhatla et al ., ; Yang et al ., ; Zhang et al ., ), appears to be unique to amphiphilic copolymers as multi‐arm PEG, which was also tested, did not affect transfection. Therefore, while the degradation products of most hydrogels are generally regarded as bioinert with respect to vector activity, these studies suggest that T904/fibrin hydrogels offer a new approach to increasing the effectiveness of non‐viral gene delivery by generating soluble degradation products that can actively increase the bioactivity of released vectors.…”

“…T904 is a four‐arm, amphiphilic block copolymer composed of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). Several studies have shown that PEO/PPO block copolymers, including T904, can improve transfection efficiency of polyplex vectors through increased endocytosis, nuclear entry and transcriptional activity (Astafieva et al ., ; Prokop et al ., ; Sriadibhatla et al ., ; Yang et al ., ; Zhang et al ., ). In the present study, acrylated T904 and FgN were crosslinked in the presence of thrombin and DTT by simultaneous enzymatic and Michael‐type addition reactions.…”