Improved Cellular Delivery of Antisense Oligonucleotide for miRNA-21 Imaging In Vivo Using Cell-Penetrating Peptide-Based Nanoprobes.

Most oligonucleotides fail to enter a cell and cannot escape from endosomes after endocytosis because of their negative charge and large molecular weight. More efficient cellular delivery of oligonucleotides should be developed for the widespread implementation of antisense imaging. The purpose of this study was to construct a novel antisense nanoprobe, Tc-labeled anti-miRNA oligonucleotides/cell-penetrating peptide PepFect6 (Tc-AMO/PF6), and to evaluate its efficacy for imaging the miRNA-21 expression in A549 lung adenocarcinoma xenografts. Naked AMO and commercial Lipofectamine 2000-based nanoparticles (AMO/LIP) were used for comparison. The cellular delivery efficiency of AMO/PF6 was first investigated by laser confocal scanning microscopy using Cy5.5-labeled probes and further validated by in vivo fluorescence imaging. Then, the probes were labeled with Tc via hydrazinonicotinamide (HYNIC). The cytotoxicity assay, cellular uptake, and retention kinetics of the probes were evaluated in vitro. The biodistribution of the probes was investigated in A549 lung cancer xenografts, and SPECT imaging was performed in vivo. AMO/PF6 showed lower cytotoxicity than AMO/LIP ( < 0.05) but showed no significant difference with naked AMO. Fluorescence microscopy demonstrated more extensive and scattered signal distribution inside the A549 cells by AMO/PF6 than AMO/LIP. The labeling efficiency of Tc-AMO/PF6 was 72.6 ± 1.42%, and the specific activity was 11.6 ± 0.13 MBq/ng. The cellular uptake of Tc-PF6/AMO peaked at 12 h, with the uptake of 11.24 ± 0.12 mol/cell × 10, and the cellular retention of Tc-AMO/PF6 was 3.92 ± 0.15 mol/cell × 10 at 12 h after interrupted incubation. AMO/PF6 showed higher cellular uptake and retention than naked AMO and AMO/LIP. The biodistribution study showed that the tumor had the highest radioactivity accumulation, with the uptake ratio of tumor/muscle (T/M) increasing from 14.59 ± 0.67 to 21.76 ± 0.98 between 1 and 6 h after injection, followed by the uptake in the kidneys and the liver. The results of in vivo fluorescence and SPECT imaging were consistent with the results of the biodistribution. The tumor was visualized at 6 h after injection of AMO/PF6 with the highest T/M ratio among these probes ( < 0.05). PF6 improves cellular delivery of antisense oligonucleotides via noncovalent nanoparticles. Tc-AMO/PF6 shows favorable imaging properties and is promising for miRNAs imaging in vivo.