Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection

Abstract

One contribution of 10 to a Theme Issue 'Molecular-, nano-and micro-devices for real-time in vivo sensing'. 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-YR 9 . oligodeoxynucleotide (ODN) hybrid and a cationic shell-crosslinked knedel-like nanoparticle (cSCK). The Y (tyrosine) residue was used for 123 I radiolabelling, whereas the R 9 (arginine 9 ) peptide was included to facilitate cell exit of untargeted PNA. Complete binding of the antisense PNA-YR 9 . 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-YR 9 . ODN . cSCK nanocomplexes efficiently entered RAW264.7 cells, whereas the PNA-YR 9 . ODN alone was not taken up. Low concentrations of 123 I-labelled antisense PNA-YR 9 . ODN complexed with cSCK showed significantly higher retention of radioactivity when iNOS was induced in lipopolysaccharide þ interferon-g-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.

Nanotechnology for delivery of peptide nucleic acids (PNAs)

“…Another PNA delivery approach uses cationic shell cross-linked knedel-like nanoparticles (cSCK) [35–38]. cSCK nanoparticles consist of a hydrophobic core and a positively charged highly functionalizable cross-linked shell.…”

“…The potential in vitro and in vivo applications of cSCK nanoparticles for PNA delivery were demonstrated using inducible nitric oxide synthase (iNOS) mRNA targeting for improved diagnostic and treatment strategies for acute lung injury (ALI) [35]. ALI is a life threatening medical condition characterized by widespread inflammation in the lungs [119].…”

“…An iNOS imaging probe was prepared by electrostatic complexation between a radiolabelled antisense PNA-YR 9. . oligonucleotide (ODN) hybrid and cSCK nanoparticle [35]. The PNA was labeled with 123 I and an R 9 (arginine 9 ) peptide was used to facilitate the exit of untargeted PNA from the cell.…”

Imaging Genetic Information

“…It is also possible that the DNA quenching strand or the quencher was being degraded in the cell. The cationic cScK NP delivery system was also investigated for its ability to image iNOS mRNA in an injured lung mouse with a radioactively labeled PNA•ODN duplex [147]. In this case, the PNA was linked to a TyrArg 9 peptide, the tyrosine of which was used to introduce radioactive 123 I and the Arg 9 of which was a cell-penetrating peptide used to facilitate exit of unbound PNA from the cell.…”

Opening windows on new biology and disease mechanisms: development of real-time in vivo sensors

“…In addition, nanoparticles have been used as dynamic and specific contrast agents for cellular magnetic resonance imaging of cancer [36]. Excitingly, Shen et al [37] report in this Theme Issue on the development of a functionalized nanocomplex to dynamically detect mRNA in vivo, a technique with implications for both normal biology and disease. Although nanoparticles will doubtlessly play a central role in the development of future biosensor technologies because of their unique properties and versatility, the long-term safety of these nanoparticles is still a controversial and emerging issue that must be addressed for effective translation of biosensors based on these technologies [38].…”