Amphiphilic block copolymers enhance cellular uptake and nuclear entry of polyplex-delivered DNA.

This work for the first time demonstrates that synthetic polymers enhance uptake and nuclear import of plasmid DNA (pDNA) through the activation of cellular trafficking machinery. Nonionic block copolymers of poly(ethylene oxide) and poly(propylene oxide), Pluronics, are widely used as excipients in pharmaceutics. We previously demonstrated that Pluronics increase the phosphorylation of IkappaB and subsequent NFkappaB nuclear localization as well as upregulate numerous NFkappaB-related genes. In this study, we show that Pluronics enhance gene transfer by pDNA/polycation complexes ("polyplexes") in a promoter-dependent fashion. Addition of Pluronic P123 or P85 to polyethyleneimine-based polyplexes had little effect on polyplex particle size but significantly enhanced pDNA cellular uptake, nuclear translocation, and gene expression in several cell lines. When added to polyplex-transfected cells after transfection, Pluronics enhanced nuclear import of pDNA containing NFkappaB binding sites, but have no effect on import of pDNA without these sites. Altogether, our studies suggest that Pluronics rapidly activate NFkappaB, which binds cytosolic pDNA that possesses promoters containing NFkappaB binding sites and consequently increase nuclear import of pDNA through NFkappaB nuclear translocation.

Transcriptional activation of gene expression by pluronic block copolymers in stably and transiently transfected cells.

Amphiphilic block copolymers of poly(ethylene oxide) and poly(propylene oxide) (Pluronics) enhance gene expression, but the mechanism remains unclear. We examined the effects of Pluronics on gene expression in murine cell models (NIH3T3 fibroblasts, C2C12 myoblasts, and Cl66 mammary adenocarcinoma cells) transfected with luciferase and green fluorescent protein. Addition of Pluronics to stably or transiently transfected cells enhanced transcription of the reporter genes. mRNA levels of the heat-shock protein hsp68 were also increased, whereas a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, was unaffected. Fibroblast and myoblast cells transfected with PathDetect cis-Reporting System constructs were used to examine the involvement of the nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) in Pluronics enhancement. Pluronics enhanced reporter gene expression controlled by NF-kappaB in both cell models. They also increased expression of a gene under AP-1 in a fibroblast cell line, but not in a myoblast cell line. Activation of the inflammation signaling pathway in myoblast cells by Pluronics was shown by increased IkappaB phosphorylation. No cytotoxicity was observed at doses of Pluronics at which gene expression was increased. Overall, these results indicate that Pluronics can increase the transcription of genes, in part, through the activation of selected stress signaling pathways.

Promoter- and strain-selective enhancement of gene expression in a mouse skeletal muscle by a polymer excipient Pluronic P85.

Amphiphilic triblock copolymers of ethylene oxide and propylene oxide (Pluronic) significantly enhanced expression of plasmid DNA in the skeletal muscle. In the presence of Pluronic P85 (P85) high levels of expression of a reporter gene (luciferase) were sustained for at least 40 days and the area under the gene expression curve increased by at least 10 times compared to the DNA alone. The effect of Pluronic depended on the strain of the mouse and the type of the promoter used. Thus, P85 enhanced luciferase expression by 17 to 19-fold in immunocompetent C57Bl/6 and Balb/c mice, while no enhancement was observed with athymic Balb/c nu/nu mice. Furthermore, P85 activated the expression of luciferase gene driven by CMV promoter, NFkappaB and p53 response elements. There was much less or no effect on the gene driven by SV40 promoter or AP1 and CRE response elements. Overall, the promoter selectivity suggested that Pluronic induced transcriptional activation of gene expression by activating the p53 and NFkappaB signaling pathways. In addition Pluronic increased the number of DNA copies and thus affected initial stages of gene transfer in a promoter selective manner.

Polymer genomics: shifting the gene and drug delivery paradigms.

Pluronic, the A-B-A amphiphilic block copolymers of poly(ethylene oxide) and poly(propylene oxide), can up-regulate the expression of selected genes in cells and alter genetic responses to antineoplastic agents in cancer. Two key new findings are discussed in relation to current drug and gene delivery strategies. First, these block copolymers alone and in combination with a polycation, polyethyleneimine, can up-regulate the expression of reporter genes in stably transfected cells. This underscores the ability of selected synthetic polymers to enhance transgene expression through a mechanism that augments improved DNA delivery into a cell. Second, although, when used alone, Pluronic is "genetically benign," when combined with an antineoplastic agent, doxorubicin, it drastically alters pharmacogenomic responses to this agent and prevents the development of multidrug resistance in breast cancer cells. Collectively, these studies propose the need for a thorough assessment of pharmacogenomic effects of polymer therapeutics to maximize the clinical outcomes and understand the pharmacological and toxicological effects of polymer-based drugs and delivery systems.

Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer.

Previously, we reported the evaluation of several polyplex-based gene delivery systems with respect to their effectiveness, toxicity, and cell type dependence in vitro. One system, P123-g-PEI(2K), a cationic graft block copolymer, is of particular interest as it has been demonstrated to successfully deliver genetic material to murine liver following systemic delivery [Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents. Gene Ther. 7, 126-138 (1)]. The P123-g-PEI(2K) system requires nonmodified Pluronic P123 as an excipient to stabilize the dispersion. The purpose of the current work was to more closely characterize this system, to assess the role of each component of the system to the overall transfection process. We evaluated particle size, stability, and resistance to nuclease degradation. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of prostate cancer cells (PC-3) with various individual components of the system. Nonmodified Pluronic alone did not significantly enhance DNA uptake, transgene expression, or DNase protection. Therefore, we conclude that nonmodified Pluronic acted primarily by optimizing the size of the polyplex. Furthermore, though this system displays several characteristics thought desirable of a nonviral gene delivery system, these studies did discriminate a potential limitation of this system for in vivo applications, namely, the insufficient level of protection of plasmid DNA from nuclease degradation. This may limit the effective dose delivered, as well as limiting the effective circulation time. These studies provide vital information that will guide modification of this system to enhance the current in vivo profile.