Abstract
Purpose :
Neovascularization is the major cause of vision loss in age-related macular degeneration (AMD) and other retinal diseases. Inflammatory biomarkers including vascular cell adhesion molecule 1 (VCAM-1) are overexpressed in endothelial cells correlating with AMD progression. In vivo imaging of VCAM-1 mRNA may allow for the detection of early pathogenic events, be used to monitor disease progression, or allow for assessment of therapeutic response. To achieve this goal, we have developed hairpin DNA-functionalized gold nanoparticle (hAuNP) probes for the in vivo detection of VCAM-1 mRNA expression in ocular tissues.
Methods :
Self-complementary, energy minimized DNA-oligonucleotides were computationally designed to achieve hairpin loop secondary structures. The optimized DNA-oligonucleotides were each coupled to a near-infrared (NIR) dye and conjugated to spherical gold nanoparticles (hAuNP). To test the utility of the new hAuNP probes, adult C57B/6 mice received laser wounds to induce choroidal neovascularization (LCNV). The VCAM-1 mRNA expression was assayed at days 0, 1, 2, 3, 6, 10 and 14 post-laser injury by qRT-PCR. The hAuNP probes were injected systemically or intraocularly in LCNV mice. In vivo imaging was performed using the Micron IV optical imaging system. The tissues were also examined ex vivo for specificity of the hAuNP probe. Scrambled oligonucleotide sequences were used as nonspecific control probes.
Results :
VCAM-1 mRNA expression peaked at day 3 post-laser as determined by qRT-PCR analysis. At this time point, hAuNP-dependent fluorescence enhancement clearly visualized the LCNV lesions with >30:1 signal to noise ratios as compared to adjacent non-injured tissues. VCAM-1 intravascular hAuNP-dependent fluorescence signal was minimal. The scrambled hAuNP probes were undetectable under the same image acquisition conditions.
Conclusions :
We have developed a targeted imaging probe for the detection of mRNA expression in real time. In vivo imaging using this hAuNP probe in LCNV mice allowed high-resolution detection of VCAM-1 mRNA expression localized to LCNV lesions. These probes may be easily adapted to transfection, knockdown or visualization of retinal gene expression in real time. The last of these has particularly strong implications for clinical application.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.