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In an effort to improve the efficacy of antisense delivery, we evaluated polyethyleneimine (PEI, 2 kDa) alone or grafted with nonionic amphiphilic block copolymer Pluronic® (P85) as a carrier for Ku86 antisense oligonucleotide (ASO) delivery. Ku86 is an abundant nuclear protein that plays an important role in nonhomologous DNA end joining and has implications in tumorigenesis and acquired drug resistance. Transfection of adherent and suspension cell lines with Ku86 ASOs complexed with P85-g-PEI (2 kDa) conjugates was associated with a specific decrease in Ku86 mRNA levels (EC50 < 75 nM and EC50 < 250 nM, respectively, n = 3). More importantly, no requirement for reduced serum conditions was necessary during transfection. In contrast, whereas Ku86 ASOs complexed with PEI (2 kDa) alone were effective in decreasing Ku86 mRNA levels in adherent cell lines (EC5O < 75 nM, n = 3), the formulation did not produce any detectable decrease in Ku86 mRNA levels in suspension cell lines. Transfection of adherent cell lines with 500 nM Ku86 ASOs formulated with P85-g-PEI (2 kDa) was associated with a specific decrease (<10% remaining of control) in Ku86 protein expression and a two-fold increased cell death after treatment with ionizing radiation (IR). In athymic nude mice bearing subcutaneous human HT29 colon adenocarcinoma xenografts, Ku86 ASO-P85-g-PEI (2 kDa) administration (15 mg/kg, subcutaneously) with a Q1D × 7 treatment schedule, when combined with a single dose of IR (6 Gy), caused a significant inhibition of HT29 tumor growth compared with mismatch- and naked antisense-pretreated control groups (time from 200 to 1000 mm³, 126.9 versus 84.18 and 87.76 days, P < 0.005). A potentiation of the antitumor activity was observed in all mice treated with Ku86 ASO-P85-g-PEI (2 kDa) formulation; however, tumor growth inhibition was reversible upon treatment cessation. No morbidity/mortality or changes in histopathology were observed under this treatment regiment. Our results indicate that P85-g-PEI (2 kDa) conjugates may increase the efficacy of Ku86 ASO delivery in management of resistant malignancies, thus providing a rationale for their evaluation in cancer patients in combination with conventional anticancer therapies. [ABSTRACT FROM AUTHOR]

DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO(26)-PO(40)-EO(26). Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DCs) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c+ CD86+, CD11c+ CD80 +, and CD11c+ CD40+. We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs.

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.

This paper, for the first time, demonstrates that exposure of cells to the poly(ethylene oxide)-poly(propylene oxide) block copolymer, Pluronic P85, results in a substantial decrease in ATP levels selectively in MDR cells. Cells expressing high levels of functional P-glycoprotein (MCF-7/ADR, KBv; LLC-MDR1; Caco-2, bovine brain microvessel endothelial cells [BBMECs]) are highly responsive to Pluronic treatment, while cells with low levels of P-glycoprotein expression (MCF-7, KB, LLC-PK1, human umbilical vein endothelial cells [HUVECs] C2C12 myoblasts) are much less responsive to such treatment. Cytotoxicity studies suggest that Pluronic acts as a chemosensitizer and potentiates cytotoxic effects of doxorubicin in MDR cells. The ability of Pluronic to inhibit P-glycoprotein and sensitize MDR cells appears to be a result of ATP depletion. Because many mechanisms of drug resistance are energy dependent, a successful strategy for treating MDR cancer could be based on selective energy depletion in MDR cells. Therefore, the finding of the energy-depleting effects of Pluronic P85, in combination with its sensitization effects is of considerable theoretical and practical significance.

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO-PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.