Imaging Dendrimer-Grafted Graphene Oxide Mediated Anti-miR-21 Delivery With an Activatable Luciferase Reporter

Abstract

MicroRNAs (miRNAs) are a class of post-transcriptional gene regulators involved in various physiological processes including carcinogenesis, and they have emerged as potential targets for tumor theranostics. However, the employment of antisense oligonucleotides, termed anti-miRs, for antagonizing miRNA functions in vivo has largely been impeded by a lack of effective delivery carriers. Here, we describe the development of polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG)-functionalized nanographene oxide (NGO) conjugate (NGO-PEG-dendrimer) for the efficient delivery of anti-miR-21 into non-small-cell lung cancer cells. To monitor the delivery of anti-miR-21 into cells and tumors, we also constructed an activatable luciferase reporter (Fluc-3xPS) containing three perfectly complementary sequences against miR-21 in the 3' untranslated region (UTR) of the reporter. Compared with bare dendrimer and Lipofectamine 2000 (Lipo2000), NGO-PEG-dendrimer showed considerably lower cytotoxicity and higher transfection efficiency. As demonstrated by in vitro bioluminescence imaging and Western blotting assays, NGO-PEG-dendrimer effectively delivered anti-miR-21 into the cytoplasm and resulted in the upregulation of luciferase intensity and PTEN target protein expression in a dose-dependent manner. Moreover, transfection with anti-miR-21 by NGO-PEG-dendrimer led to stronger inhibition of cell migration and invasion than did bare dendrimer or Lipo2000 transfection. The intravenous delivery of anti-miR-21 via NGO-PEG-dendrimer induced a significant increase in the bioluminescence signal within the Fluc-3xPS reporter-transplanted tumor areas. These results suggest that NGO-PEG-dendrimer could be an efficient and a potential nanocarrier for delivering RNA oligonucleotides. In addition, the strategy of combining NGO-PEG-dendrimer with an activatable luciferase reporter allows the image-guided monitoring of the delivery process, which can provide insights into the RNA-based cancer treatments.

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…The miRNAs (miR15a 11 , miR155 11 and miR-21 5 ) in different tumour environments can be detected by enhanced LNA FISH probes with 11 or without 5 amplification methods. The miR22 67 from A549 cells and miR16 74 from chemoresistant SGC7901/VCR cells as well as miR16 74 from xenografts were detected by reporter gene systems (Fig. 17).…”

“…Ko et al applied a CMV/Gluc/3×PT_mi124a reporter gene system 71 , illustrating the miR124a-dependent decrease of Gluc reporter activity during neuronal differentiation 70 . Wang et al developed a CMV/Fluc/3×TS_miR21 reporter system to image anti-miR21 67 . Once exogenous anti-miR-21 oligonucleotides carried by GO-PEG-dendrimer enter the cells, they inhibit the functions of miR-21, and therefore block miR-21 from binding to the 3′-UTR of luciferase, activating the luciferase reporter and inducing bioluminescence 67 .…”

“…Wang et al developed a CMV/Fluc/3×TS_miR21 reporter system to image anti-miR21 67 . Once exogenous anti-miR-21 oligonucleotides carried by GO-PEG-dendrimer enter the cells, they inhibit the functions of miR-21, and therefore block miR-21 from binding to the 3′-UTR of luciferase, activating the luciferase reporter and inducing bioluminescence 67 .…”

Recent progress in microRNA-based delivery systems for the treatment of human disease

“…Recently, Wang et al demonstrated a novel carrier denoted as NGO-PEG-dendrimers for miRNA delivery. NGO-PEG-dendrimers/anti-miRNA-21 were fabricated by conjugating PAMAM dendrimers and PEG-functionalized nanographene oxide (NGO) to 2′-O-methyl-modified anti-miRNA-21 [95]. The intravenous injection of NGO-PEG-dendrimers/anti-miRNA-21 caused a remarkable increase in bioluminescence signals within tumor areas through a luciferase reporter [95].…”

Polymer nanocarriers for MicroRNA delivery

“…When complexed with biological molecules, dendrimers are called dendriplexes, and cationic dendrimers are a practical choice of carrier for miRNA because they can readily associate with negatively charged nucleic acids. While many dendrimer types have been explored for siRNA delivery and antagomiR delivery, little research has examined the use of dendrimers for miRNA mimic delivery. Below, we describe some of the findings in this field to date.…”