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.

Keywords: Gold nanoparticle; Imaging; Microvascular endothelial cells; VCAM-1; mRNA.

Figure 1

Structural design, stability, sensitivity and hybridization kinetics of hAuNP. (A) Gold nanoparticles were functionalized with DNA hairpin-loops incorporating anti-sense sequence specific for mouse VCAM-1 mRNA (AS-VCAM-1 hAuNP) or a scrambled version of this sequence (NS hAuNP). DNA hairpin-loops are modified on their 5′ ends with a thiol (SH) group and coupled to gold nanoparticles through an Au-S bond. In the absence of VCAM-1 mRNA, the stem region (green) maintains the hairpin structure through self-complementary hydrogen bonding. The hairpin structure requires the 3′ near-infrared (NIR) fluorophore to be held in close proximity to the gold surface. The capacity of gold to quench fluorescence is maximal in this configuration; therefore, no fluorescence is observed. Hybridization of a target mRNA to the anti-sense recognition sequence (blue) causes the hairpin to open, increasing the distance between the NIR dye and the gold surface, and resulting in fluorescence emission. (B) AS-VCAM-1 hAuNP are stable in a variety of media. Fluorescence was only observed when a complimentary single strand (ss) DNA oligonucleotide (VCAM-1 oligonucleotide) was present, indicating the specificity of the AS-VCAM-1 hAuNP. (C) The hybridization kinetics of the AS-VCAM-1 hAuNP in the presence of the VCAM-1 oligonucleotide depended on the reaction medium. These studies also confirmed the specificity of the AS-VCAM-1 hAuNP for the exogenous VCAM-1 oligonucleotide compared to the scrambled sequence (NS oligonucleotide). (D) AS-VCAM-1 hAuNP exposed to increasing doses of the VCAM-1 oligonucleotide exhibited a dose-dependent increase in fluorescence. The same effect was not observed when AS-VCAM-1 hAuNP was exposed to the NS oligonucleotide.

Figure 2

Stability of AS-VCAM-1 hAuNP and citrate-capped GNP (CT-GNP) in different media. (A) The spectroscopic analysis of AS-VCAM-1 hAuNP and CT-GNP. The 520 nm absorbance maximum was stable for AS-VCAM-1 hAuNP in PBS and EBM indicating monodispersions. The CT-GNP exhibits an absorbance profile with a maximum 520nm in water. However, in PBS and EBM this profile is significantly altered, presumably due to aggregation. (B) Qualitative analysis of CT-GNP in different media, indicating a monodispersion in water (red). A color shift to blue/violet was observed in isotonic media, thus indicating aggregation of the CT-GNP. (C) Qualitative analysis of AS-VCAM-1 hAuNP indicated their stability (no aggregation) in each of these media (all red). (D) Transmission electron microscopy (TEM) micrograph of CT-GNP showing monodispersion in water, and aggregation in PBS and cell culture medium, consistent with A and B.

Figure 3

Specificity of AS-VCAM-1 hAuNP for VCAM-1 mRNA. (A) Total RNA was isolated from TNF-α- (10 ng/ml) treated MRMEC at 1 to 24 hours. VCAM-1 mRNA expression levels relative to 18s was determined by qRT-PCR. Expression values are expressed as fold change relative to unstimulated cells. Maximum expression was observed at 4 hours that was increased by approximately 75-fold over the control. (B) Total RNA was isolated from MRMEC treated with TNF-α (10 ng/ml) or vehicle. Hybridization reactions were performed by incubating the total RNA samples with AS-VCAM-1 hAuNP or NS hAuNP at 37°C for 2 hours. A 6.25-fold statistically significant signal enhancement was observed in reactions with AS-VCAM-1 hAuNP compared to those with NS hAuNP. The results of this experiment indicates the ability of AS-VCAM-1 hAuNP to discriminate VCAM-1 mRNA against the TNF-α-induced MRMEC transcriptome.

Figure 4

Transmission electron microscopy (TEM) imaging of MRMECs and detection of intracellular AS-VCAM-1 hAuNP. (A and B) TEM micrograph of MRMECs cultured in normal growth medium plus AS-VCAM-1 hAuNP. (A) Clusters of hAuNPs were visible on the surface of the plasma membrane (red arrow). (B) intracellular hAuNP were observed just beneath the plasma membrane (yellow arrow) and further into the cell’s interior (white arrow). (C, D and E) TNF-α treated MRMECs incubated with AS-VCAM-1 hAuNP. (C) Clusters of AS-VCAM-1 hAuNP were localized in endosomes or lysosomes throughout the cytoplasm as indicated by red and yellow arrows. Panel D is an inset from C, the red arrows indicate AS-VCAM-1 hAuNP internalized in a lysosome/endosome. Panel E is another inset from C; the yellow arrows indicate the AS-VCAM-1 hAuNPs internalized in multivesicular bodies in the cytoplasm. AS-VCAM-1- and NS hAuNP were indiscriminately internalized by MRMEC independent of treatment.

Figure 5

Confocal imaging of live MRMECs treated with AS-VCAM-1 hAuNP and NS hAuNP. Cells were cultured on microscope slides and treated with TNF-α or vehicle plus AS-VCAM-1 hAuNP or NS hAuNP in complete growth medium. After a 6-hour incubation, these media were aspirated and fresh medium was added to each culture. The cells were imaged using confocal microscopy. (A) Strong fluorescence emission was only detected in TNF-α-activated MRMEC treated with AS-VCAM-1 hAuNP. (B, C and D) Only minimal fluorescence was detected in the other cultures. (E) Fluorescence intensity is expressed in relative fluorescence units (RFU) per cell over different treatment groups as measured using ImageJ software.

Figure 6

Cytotoxicity of AS-VCAM-1 hAuNP, NS hAuNP and CT-GNP. Cytotoxicity was measured by the live-dead assay using Calcein AM. AS-VCAM-1 and NS hAuNP did not significantly reduce MRMEC viability at 1 and 5 nM when compared to normal serum-treated cells. Citrate-capped 15nm gold nanoparticle (CT-GNP) showed reduced cell viability compared to control (p <0.05). Data are presented as mean ± SD (n = 5).