title: Enhanced intracellular uptake and inhibition of NF-kappaB activation by decoy oligonucleotide released from PLGA microspheres.
abstract: BACKGROUND
  Nuclear factor-kappaB (NF-kappaB) transcription factor regulates the expression of genes involved in immune response and inflammation. NF-kappaB activity can be efficiently inhibited by double-stranded oligodeoxynucleotides (ODNs). In the present study, we investigated the potential of poly(DL-lactic-co-glycolic acid) (PLGA) microspheres as delivery system for an ODN against NF-kappaB in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS).
  
  
  METHODS
  Microspheres encapsulating ODN were prepared by the multiple emulsion/solvent evaporation technique and characterised in terms of size, morphology, encapsulation efficiency and in vitro release profile. In vitro uptake after 4 h and activity of ODN released from microspheres were evaluated in RAW 264.7 macrophages stimulated with LPS for 24, 48 and 72 h.
  
  
  RESULTS
  We prepared microspheres with a high encapsulation efficiency showing a very slow and almost constant in vitro release of ODN for up to 1 month. ODN slowly released from microspheres translocated better into LPS-stimulated cells as compared with naked ODN. Incubation of cells with ODN-encapsulating microspheres resulted in a decrease of tumor necrosis factor-alpha (TNF-alpha) and nitrite production, inducible nitric oxide synthase (iNOS) protein expression, as well as NF-kappaB/DNA-binding activity. Similar results were obtained with naked ODN only at about 80 times higher concentrations.
  
  
  CONCLUSIONS
  Our results suggest that PLGA microspheres could be a useful tool to improve pharmacokinetics of a ODN decoy to NF-kappaB and may represent a promising strategy to effectively inhibit the transcriptional activity of NF-kappaB in inflammatory process.
authors.affiliation: Dipartimento di Chimica Farmaceutica e Tossicologica, Università degli Studi di Napoli Federico II, Via Montesano 49, 80131 Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: De Rosa
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Maiuri
authors.forename: Francesca
authors.initials: F
authors.lastname: Ungaro
authors.forename: Daniela
authors.initials: D
authors.lastname: De Stefano
authors.forename: Fabiana
authors.initials: F
authors.lastname: Quaglia
authors.forename: Maria Immacolata
authors.initials: MI
authors.lastname: La Rotonda
authors.forename: Rosa
authors.initials: R
authors.lastname: Carnuccio
mesh_terms: D000818:Animals; D002460:Cell Line; D003692:Delayed-Action Preparations / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D019344:Lactic Acid / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers / Q000737:chemistry*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D003692:Delayed-Action Preparations; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D009841:Oligonucleotides; D011108:Polymers; D014409:Tumor Necrosis Factor-alpha; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse