title: Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection.
abstract: Acute lung injury (ALI) is a complex syndrome with many aetiologies, resulting in the upregulation of inflammatory mediators in the host, followed by dyspnoea, hypoxemia and pulmonary oedema. A central mediator is inducible nitric oxide synthase (iNOS) that drives the production of NO and continued inflammation. Thus, it is useful to have diagnostic and therapeutic agents for targeting iNOS expression. One general approach is to target the precursor iNOS mRNA with antisense nucleic acids. Peptide nucleic acids (PNAs) have many advantages that make them an ideal platform for development of antisense theranostic agents. Their membrane impermeability, however, limits biological applications. Here, we report the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabelled antisense PNA-YR9 · oligodeoxynucleotide (ODN) hybrid and a cationic shell-cross-linked knedel-like nanoparticle (cSCK). The Y (tyrosine) residue was used for (123)I radiolabelling, whereas the R9 (arginine9) peptide was included to facilitate cell exit of untargeted PNA. Complete binding of the antisense PNA-YR9 · ODN hybrid to the cSCK was achieved at an 8 : 1 cSCK amine to ODN phosphate (N/P) ratio by a gel retardation assay. The antisense PNA-YR9 · ODN · cSCK nanocomplexes efficiently entered RAW264.7 cells, whereas the PNA-YR9 · ODN alone was not taken up. Low concentrations of (123)I-labelled antisense PNA-YR9 · ODN complexed with cSCK showed significantly higher retention of radioactivity when iNOS was induced in lipopolysaccharide+interferon-γ-activated RAW264.7 cells when compared with a mismatched PNA. Moreover, statistically, greater retention of radioactivity from the antisense complex was also observed in vivo in an iNOS-induced mouse lung after intratracheal administration of the nanocomplexes. This study demonstrates the specificity and sensitivity by which the radiolabelled nanocomplexes can detect iNOS mRNA in vitro and in vivo and their potential for early diagnosis of ALI.
authors.affiliation: Department of Chemistry , Washington University , St Louis, MO 63130 , USA.
authors.forename: Yuefei
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Chemistry and Chemical Engineering , Texas A&M University , PO Box 30012, College Station, TX 77842-3012 , USA.
authors.forename: Ritu
authors.initials: R
authors.lastname: Shrestha
authors.affiliation: Department of Medicine , Washington University , St Louis, MO 63110 , USA.
authors.forename: Aida
authors.initials: A
authors.lastname: Ibricevic
authors.affiliation: Department of Medicine , Washington University , St Louis, MO 63110 , USA.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Gunsten
authors.affiliation: Department of Radiology , Washington University , St Louis, MO 63110 , USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welch
authors.affiliation: Department of Chemistry and Chemical Engineering , Texas A&M University , PO Box 30012, College Station, TX 77842-3012 , USA.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Wooley
authors.affiliation: Department of Medicine , Washington University , St Louis, MO 63110 , USA ; Department of Radiology , Washington University , St Louis, MO 63110 , USA.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Brody
authors.affiliation: Department of Chemistry , Washington University , St Louis, MO 63130 , USA.
authors.forename: John-Stephen A
authors.initials: JS
authors.lastname: Taylor
authors.affiliation: Department of Radiology , Washington University , St Louis, MO 63110 , USA.
authors.forename: Yongjian
authors.initials: Y
authors.lastname: Liu
keywords: acute lung injury; cationic nanoparticles; inducible nitric oxide synthase; peptide nucleic acid; radiolabelling; targeting