ABSTRACT
PURPOSE: To evaluate the effect of re-directing the uptake mechanism of polyplexes containing oligodeoxynucleotide (ODN) decoys to nuclear factor kappa B (NF-kappaB) from absorptive-mediated to receptor-mediated endocytosis. MATERIALS AND METHODS: Complexes of ODNs and a co-polymer of biotin-polyethylenglycol and polyethylenimine (BPP) were targeted to brain-derived endothelial cells with a conjugate of antibody 8D3 and streptavidin (8D3SA). Size and stability of ODN/BPP complexes was measured by dynamic light scattering. Cellular uptake was studied by confocal microscopy. Cell viability and pharmacological effects were investigated on murine bEnd5 cells stimulated with tumor necrosis factor. RESULTS: ODN/BPP complexes showed sizes of 116+/-2.3 nm, which increased by 40 nm when coupled to 8D3SA, and were stable in physiological fluids. Targeted complexes were internalized intact into endosomal compartments. Treatment conditions, which yielded significant inhibitory effects on mRNA expression of VCAM-1, ICAM-1, IkappaBalpha and iNOS by bEnd5 cells, did not affect viability. At 0.5 microM, decoy ODN significantly inhibited monocyte adhesion to bEnd5 monolayers when delivered as 8D3SA-targeted complex, while higher concentrations of untargeted complex were ineffective. CONCLUSIONS: The complex of NF-kappaB decoys and BPP, which can be targeted to transferrin receptors, is a promising drug candidate for neuroinflammatory diseases affecting the blood-brain barrier.
Subject(s)
Anti-Inflammatory Agents/metabolism , Biotin/metabolism , Brain/blood supply , Drug Carriers , Endocytosis , Endothelial Cells/metabolism , NF-kappa B/metabolism , Oligonucleotides/metabolism , Polyethylene Glycols/metabolism , Polyethyleneimine/metabolism , Receptors, Transferrin/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Biotin/toxicity , Cell Adhesion , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Line, Transformed , Cell Survival , Dose-Response Relationship, Drug , Down-Regulation , Endosomes/metabolism , Endothelial Cells/drug effects , Mice , NF-kappa B/genetics , Oligonucleotides/pharmacology , Particle Size , Polyethylene Glycols/toxicity , Polyethyleneimine/toxicity , RNA, Messenger/metabolism , Time Factors , Transcription, Genetic , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: The nuclear factor (NF)-kappaB plays a key role in inflammatory reactions of the endothelium by controlling the expression of surface-adhesion molecules and other inflammatory mediators, which facilitate the attachment of monocytes and lymphocytes to the endothelial surface. We investigated the inhibition of monocyte adhesion by NF-kappaB transcription factor decoys complexed with polyethylenimines (PEIs) of different molecular weights and structures (800, 25, and 2.7 kDa PEI). METHODS: Formation, size and stability of the PEI/decoy complexes were investigated by polyacrylamide gel electrophoresis and photon correlation spectroscopy. The efficiency of the complexes was studied in a cell adhesion assay using the murine brain-derived endothelial cell line bEnd5, activated with lipopolysaccharide as inflammatory model. U-937 monocytes were fluorescently labeled with BCECF-AM to permit quantitative measurement of adhesion. Expression of endothelial cell adhesion molecules was determined at the mRNA level by RT-PCR and at the protein level by ELISA. RESULTS: Depending on the N/P ratio, decoys formed complexes of <200 nm in size with all PEIs, which were stable against degradation by nucleases and dissociation by albumin. Treatment of bEnd5 and U-937 cells with NF-kappaB decoys complexed with 25 and 2.7 kDa PEI reduced the number of adherent U-937 cells and decreased the levels of ICAM-1 and VCAM-1 mRNA and protein. The effects were specific, time-dependent and increased with higher N/P ratios of complexes and lower cytotoxicity of polymers. In contrast, the efficiency of the 800 kDa PEI was much lower compared to the other polymers. CONCLUSIONS: Complexes of NF-kappaB decoy and PEIs effectively inhibited the adherence of monocytes on endothelial cells, which could be a promising strategy for the treatment of inflammatory diseases.
Subject(s)
Brain/cytology , Endothelial Cells/cytology , Monocytes/cytology , Monocytes/drug effects , Oligodeoxyribonucleotides/pharmacology , Polyethyleneimine/pharmacology , Animals , Cell Adhesion/drug effects , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Survival , Cells, Cultured , Down-Regulation/drug effects , Down-Regulation/genetics , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , In Vitro Techniques , Mice , Monocytes/metabolism , Oligodeoxyribonucleotides/chemistry , Polyethyleneimine/chemistry , U937 CellsABSTRACT
The in vivo body distribution and the pharmacokinetics of a 20mer double-stranded nuclear factor kappaB decoy oligodeoxynucleotide (ODN) complexed with 25-kDa poly(ethylene imine) (PEI), low molecular weight 2.7-kDa PEI, and PEGylated PEI [bPEI(25k)-glPEG(550)(50)] after intravenous injection were studied in BALB/c mice using a double-labeling technique to follow simultaneously the distribution of both complex components. The polymers were radioactively labeled with (125)I by Bolton-Hunter reagent and the decoys with [gamma-(32)P]ATP by an enzymatic 5'-end-labeling technique. After i.v. bolus injections into the jugular vein, organ samples were taken after 15 min, 2 h and 12 h. For pharmacokinetic studies blood and plasma samples were collected from 20 s up to 2 h. Uncomplexed decoy was found to be degraded already after 15 min and was rapidly eliminated renally into urine. Complexation with the homopolymers increased the organ levels and circulation time of ODN after 15 min, with similar organ distribution profiles for (125)I and (32)P. In contrast to the behavior of free ODN, the complexes were mainly distributed into liver and spleen. Whereas the organ concentrations of (125)I remained high over 12 h, the (32)P values of ODN decreased in a time-dependent manner, likely due to separation of the complexes and degradation of the DNA. Although PEGylated PEI demonstrated a slower (125)I-uptake into the RES organs compared with 25-kDa PEI due to the shielding effect of PEG [poly(ethylene glycol)], it was not able to better stabilize the complexes in the circulation or protect DNA from degradation.
