In vivo messenger RNA detection with antisense PNA functionalized cationic nanocomplex

1221 Objectives The goal of this study was to prepare an antisense peptide nucleic acid (PNA) functionalized molecular nanoprobe for specific determination of inducible nitric oxide synthase (iNOS) up-regulated in an acutely injured lung model. Methods The cationic nanocomplex of 123I-PNAantiYR9-ODNanti-cSCK is formulated by electrostatic complexation of a cSCK and a radiolabeled antisense 123I-PNAantiYR9-ODNanti hybrid with high binding affinity to iNOS mRNA (Nos2). The intracellular localization of the nanocomplex was tracked through a fluorescent dye. The cSCK amine to ODN phosphate ratio (N/P) was optimized to minimize the cytoxicity. The in vitro binding specificity of developed anti-sense nanocomplex was tested in lipopolysaccharide (LPS) and interferon-gamma stimulated RAW 264.7 cells expressing high concentration of iNOS and compared to the sense PNA constructed nanocompelex following same strategy. In vivo targeting specificity of the nanocomplexes was demonstrated through biodistribution in a LPS injured mouse lung model. Results Complete binding of the antisense PNA-YR9-ODN hybrid to the cSCK was achieved at an 8:1 N/P ratio by a gel retardation assay. The antisense PNA-YR9-ODN-cSCK nanocomplexes efficiently entered RAW cells, while the PNA-YR9-ODN alone was not taken up. Low concentrations of 123I-PNAantiYR9-ODNanti-cSCK showed significantly higher retention of radioactivity in iNOS up-regulated RAW cells when compared to a sense PNA nanocomplex. Statistically greater retention of radioactivity from 123I-PNAantiYR9-ODNanti-cSCK was also observed in an iNOS-induced mouse lung after intratracheal administration. Conclusions This work demonstrates the specificity and sensitivity of the 123I-PNAantiYR9-ODNanti-cSCK nanocomplex for in vitro and in vivo iNOS mRNA detection and its potential for early diagnosis of acute lung injury. Research Support This work is supported by the NHLBI Program of Excellence in Nanotechnology (HHSN268201000046C).