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Title

Development of Antisense Therapeutic and Imaging Agents to Detect and Suppress Inducible Nitric Oxide Synthase (iNOS) Expression in Acute Lung Injury (ALI)

Date of Award

Spring 5-15-2013

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

This dissertation focuses on the development and investigation of antisense imaging and therapeutic agents, combined with nanotechnology, to detect and suppress inducible nitric oxide synthase (iNOS) expression for the diagnosis and treatment of acute lung injury (ALI). To achieve this goal, several efforts were made.

The first effort was the identification and characterization of high binding affinity antisense peptide nucleic acids (PNAs) and shell-crosslinked knedel-like nanoparticle (SCK)-PNA conjugates to the iNOS mRNA. Antisense binding sites on the iNOS mRNA were first mapped by a procedure for rapidly generating a library of antisense accessible sites on native mRNAs (MASL) which involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific PCR. Antisense PNAs against the antisense accessible sites were accordingly synthesized and characterized. The dissociation constants of oligodeoxynucleotides (ODNs) to iNOS mRNA were quantitatively measured by a Dynabead-based assay, and the binding affinities of PNAs were determined by a competition assay. All the sites identified by the MASL assay showed high binding affinities with pM dissociation constants. Finally, hierarchically-assembled, multi-functional SCKs were developed and conjugated with PNAs to promote delivery and recognition. The PNA-SCK conjugates also showed sequence-specific binding to iNOS mRNA by comparing the binding affinity of matched and mismatched PNA-SCK conjugates.

The second effort was the investigation of cationic shell crosslinked knedel-like nanoparticle (cSCK)-mediated siRNA delivery to suppress iNOS expression for the treatment of ALI. siRNA with its unique gene-specific properties could serve as a promising therapeutic agent, however success in this area has been challenged by a lack of efficient biocompatible transfection agents. cSCK with its nanometer size and positive charge previously showed efficient cellular delivery of phosphorothioate ODNs (oligodeoxynucleotides), plasmid DNA and PNA. Herein, cSCK showed good siRNA binding and facilitated efficient siRNA transfection in HeLa, a mouse macrophage cell line and other human cell lines. cSCK led to greater silencing efficiency than Lipofectamine 2000 in HeLa cells as determined by the viability following transfection with cytotoxic and non-cytotoxic siRNAs, as well in 293T and HEK cells, and was comparable in BEAS-2B and MCF10a cells. cSCK also led to greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.

The third effort was the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabeled antisense PNA-YR9*oligodeoxynucleotide (ODN) hybrid and a cationic shell-crosslinked knedel-like nanoparticle (cSCK) to specifically target and image iNOS mRNA toward the diagnosis of ALI. The Y (tyrosine) residue was used for 123I radiolabeling while the R9 (arginine9) peptide was used to facilitate endosomal, lysosomal, and cellular escape of untargeted PNA probe. 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. The antisense PNA-YR9*ODN*cSCK nanocomplexes efficiently entered RAW 264.7 cells, while the PNA-YR9*ODN alone was not taken up. Low concentrations of 123I-labeled antisense PNA-YR9*ODN complexed with cSCK, showed significantly higher retention of radioactivity in iNOS-induced RAW 264.7 cells when compared to 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 work demonstrates the specificity and sensitivity by which the radiolabeled nanocomplexes can detect iNOS mRNA in vitro and in vivo and their potential for early diagnosis of acute lung injury.

The fourth effort was the study of a degradable polyphosphoester-based cationic nanoparticle (dg-cSCK), which itself demonstrated efficient iNOS inhibition without further loading of any other therapeutic drugs. It appeared that spontaneous hydrolytic degradation and/or assisted by enzymes caused the particle to quickly release degraded small fragments. One of the expected degradation products showed dose-dependent iNOS inhibition, and might serve as a novel inhibitor that could explain the behavior of dg-cSCK. Dg-cSCK showed much more efficient iNOS inhibition than the degradation product, probably due to higher cellular uptake on the nanoparticle precursor than the degradation product. Dg-cSCK also led to the decrease of iNOS mRNA level, suggesting that the inhibition might be taking place upstream of iNOS. This degradable PPE-based cationic nanoparticle might have potential as a promising anti-iNOS and/or anti-inflammatory agent for the treatment of acute lung injury. Future work will focus on identifying upstream members of the iNOS pathway that may be inhibited by dg-cSCK, and the application of this nanoparticle to ALI animal models.

Language

English (en)

Chair and Committee

John-Stephen Taylor

Committee Members

Robert Blankenship, Steven Brody, Michael Gross, Yongjian Liu, Joshua Maurer

Comments

Permanent URL: https://doi.org/10.7936/K7RJ4GCT

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