title: Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells.
abstract: High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG2000 chains (PHEA-PEG-Spm). PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350-650 nm and 100-400 nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42-47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells. In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo studies are warranted.
authors.affiliation: Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy.
authors.forename: Sante
authors.initials: S
authors.lastname: Di Gioia
authors.affiliation: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo, 90123 Palermo, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Sardo
authors.affiliation: Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Belgiovine
authors.affiliation: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo, 90123 Palermo, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Triolo
authors.affiliation: Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: d'Apolito
authors.affiliation: Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Castellani
authors.affiliation: Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy; Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Medical Genetics Laboratory, 20132 Milan, Italy.
authors.forename: Annalucia
authors.initials: A
authors.lastname: Carbone
authors.affiliation: Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy.
authors.forename: Ida
authors.initials: I
authors.lastname: Giardino
authors.affiliation: IBF-CNR, 90143 Palermo, Italy.
authors.forename: Gaetano
authors.initials: G
authors.lastname: Giammona
authors.affiliation: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo, 90123 Palermo, Italy. Electronic address: gennara.cavallaro@unipa.it.
authors.forename: Gennara
authors.initials: G
authors.lastname: Cavallaro
authors.affiliation: Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Conese
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D060785:Mammaglobin A / Q000096:biosynthesis*; D008422:Materials Testing; D049329:Nanostructures; D010455:Peptides / Q000737:chemistry*; D011102:Polyhydroxyethyl Methacrylate / Q000031:analogs & derivatives; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000378:metabolism / Q000494:pharmacology*; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
chemical_list: D060785:Mammaglobin A; D010455:Peptides; D034741:RNA, Small Interfering; C556448:SCGB2A2 protein, human; D011102:Polyhydroxyethyl Methacrylate; C023037:poly(2-hydroxyethyl acrylate); D004247:DNA
keywords: Airway epithelial cells; HMGB1; Nucleic acid-based drugs; PHEA; Polyaspartamide copolymers; Sirna