posted on 2021-01-23, 02:00authored byGuangjie Yang, Yujun Zhao, Aidi Gong, Wenjie Miao, Lei Yan, Pei Nie, Zhenguang Wang
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, 99mTc-labeled anti-miRNA oligonucleotides/cell-penetrating
peptide PepFect6 (99mTc-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 99mTc 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 (P < 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 99mTc-AMO/PF6
was 72.6 ± 1.42%, and the specific activity was 11.6 ± 0.13
MBq/ng. The cellular uptake of 99mTc-PF6/AMO peaked at
12 h, with the uptake of 11.24 ± 0.12 mol/cell × 10–16, and the cellular retention of 99mTc-AMO/PF6
was 3.92 ± 0.15 mol/cell × 10–16 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 (P < 0.05). PF6 improves cellular delivery of antisense
oligonucleotides via noncovalent nanoparticles. 99mTc-AMO/PF6
shows favorable imaging properties and is promising for miRNAs imaging
in vivo.