Real-time imaging of VCAM-1 mRNA in TNF- α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles

Abstract

Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.

A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

“…DLS measurements were achieved by following a slightly modified protocol of our previously published method 24 . Briefly, on a Malvern Zetasizer Nano ZS (Malvern Instruments, Inc.; Westborough, MA).…”

“…TEM imaging was acquired 24 by mounting the shRNA–lipid probes on 300-mesh copper grids and stained with 2% uranyl acetate. Samples were subsequently imaged on the Philips/FEI Tecnai T12 electron microscope (Hillsboro, OR) at various magnifications.…”

“…However, most of these hybridization methods require the use of fixed tissues or endogenously labelled target mRNA for imaging and tracking. Recent development of gold-mediated targeted delivery of oligonucleotides facilitates the real-time imaging of mRNA in living cells 24 . In this current study, we have designed and synthesized AS-Eng shRNA–lipid conjugates for targeted imaging of endoglin mRNA that is associated with neovascularization in living retinas without using any toxic transfection reagents.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Others, like Xue et al, have detected oncogenes like STAT5B in MCF‐7 cells . Uddin et al have used NP‐MB for detection of VCAM‐1 mRNA, a marker of inflammation, in retinal microvascular endothelial cells . Work by Qiao et al has expanded on the original construct by conjugating two different MBs on the gold surface to do multiplexed analysis of survivin and cyclin D1 mRNA .…”

Interactions Between Tumor Biology and Targeted Nanoplatforms for Imaging Applications

“…The pattern of hypo‐intensity in the MRI images was in agreement with the distribution of the contrast agent measured by phosphor‐imaging and SPECT. Uddin et al110 used the VCAM‐1 targeted antisense hairpin and DNA‐functionalized gold NPs (AS‐VCAM‐1 hAuNP) for real time detection and imaging of VCAM‐1 expression in retinal endothelial cells. An increase in VCAM‐1 mRNA levels caused a fluorescence enhancement that was clearly visible and increased the signal/noise ratio.…”