| Objective Preparation of a novel AMO/PF6 nanoparticle probe,to study the antisense imaging value of the probe in the lung adenocarcinoma model of miRNA-21 overexpression.Methods The antisense oligonucleotide sequence of miRNA-21,AMO,labeled with Cy5.5fluorescent and the cell penetrating peptide(PF6)were synthesized and designed.PF6(stock solution 100 mM)was mixed with AMO(stock solution 10 mM)in Milli-Q water and incubated at room temperature for 30 min to get AMO/PF6 nanoprobes.The molar ratio(MR)of PF6 to AMO(final concentration of 75 nM)was 30:1 and the charge ratio was 2:1.The liposome transfection reagent(Lipofectamine 2000,LIP)was used as a control,simultaneously synthesize AMO/LIP nanoprobes.The AMO/PF6 nanoparticle probes were characterized by size and shape using transmission electron microscope(TEM)and compared with AMO/LIP nanoparticles.AMO,AMO/PF6 and AMO/LIP nanoparticles were transfected into lung adenocarcinoma A549 cells respectively.After transfection for 6 h and8 h,the transfection effect was observed by laser scanning confocal microscope(LSCM).AMO/PF6 nanoparticles cytotoxicity was detected by cell proliferation-toxicity kit(Cell Counting Kit-8,CCK8).The final concentrations of AMO were designed to be 50 nM,75 nM,100 nM and 200 nM respectively.The MRs of AMO and PF6 included 10:1,20:1 and30:1,and the optical density(OD)between the experimental group and the control group.The ratio was taken as the relative cell survival rate and the cytotoxicity of AMO,AMO/PF6,AMO/LIP under different conditions was compared.A549 lung adenocarcinoma-bearing mouse model was constructed and 10% glucose solution(corrected fluorescence imaging system),AMO-10% glucose solution,AMO/PF6-10% glucose solution,AMO/LIP-10%glucose solution were injected into the tail vein respectively.After anesthesia with 10%chloral hydrate,the distribution of fluorescence signals in vivo were measured using in vivo imaging system(IVIS)(every 30 minutes until 150 min).The region of interest of the tumor and liver was selected by Image-Pro Plus 6.0 software,and the tumor-to-liver uptake ratio(Tumor/Liver,T/L)of each group was calculated and statistically analyzed.In vitro tissueimaging and frozen section imaging: after the end of in vivo imaging,the animals were sacrificed by cervical dislocation.In vitro imaging and frozen section imaging were performed to detect whether there was a difference in the distribution of AMO in vitro and in vivo.Differences between the two groups were compared with two independent sample t-tests,and P < 0.05 was considered to be statistically significant.Result AMO/PF6 nanoprobe was successfully synthesized.TEM observation showed that AMO/PF6 nanometer probes were nearly spherical with a smooth surface and a diameter of30-40 nm.AMO/LIP nanoprobe was nearly ellipsoidal with rough surface and a diameter of50-70 nm.After transfection for 6 h and 8 h,only a small amount of fluorescence signal could be seen in A549 cells of AMO group.After 6 h transfection with AMO/LIP probe group,a large number of fluorescence signals appeared in A549 cells.And extended to 8 h,the fluorescence signals were increased and the signals were distributed in the nucleus and cytoplasm.After transfection for 6 h in AMO/PF6 probe group,A549 cells also presented a large number of fluorescence signals.And extended to 8 h,fluorescence signals were significantly increased which were more than that in AMO/LIP probe group and mainly distributed in the cytoplasm.The results of CCK8 cytotoxicity assay showed that AMO,AMO/PF6 and AMO/LIP nanoprobes had certain cytotoxicity compared with the blank group.At the same concentration,AMO had the lowest toxicity and the highest relative survival rate(50 nM group: 89.89%,75 nM group: 90.10%,100 nM group: 88.15% and 200 nM group: 87.73%).When the final concentration of AMO was 75 nM,the relative survival rate of the three groups was the highest compared with other groups(AMO group: 90.10%,AMO/PF6 group: 89.42%,AMO/LIP group: 86.33%).The relative survival rate of PF6 and AMO was the highest when the final concentration of AMO was 75 nM at MR 30:1(10:1group: 88.92%,20:1 group: 88.81% and 30:1 group: 89.42%).By comparing the cytotoxicity of AMO/PF6 probe and AMO/LIP probe,it was found that the relative cell survival rate of AMO/PF6 group was higher than that of AMO/LIP group at the same concentration regardless of the mixing ratio of AMO/PF6 probe.Under the condition of MR30:1,the difference between AMO/PF6 group and AMO/LIP group was statistically significant(50 nM group: 89.17±0.73% vs.85.98±0.23%,P=0.014;75 nM group:89.43±0.48 % vs.86.33±0.14%,P=0.004;100 nM group: 87.46±0.45% vs.81.95±0.94%,P=0.006;200 nM group: 85.31±0.47% vs.78.94±0.93%,P=0.004).After 120 min of AMO-10% glucose solution,the fluorescence signals were mainly concentrated in liver,kidney and tumor.The highest T/L ratio was 1.118±0.004.After 120 min of AMO/LIP probe injection,fluorescence signals were clustered in liver,kidney and tumor,but after 150 min of injection,fluorescence signals were clustered in lung.Fluorescence signals were concentrated in the tumor,liver and kidney,and there was no signal aggregation in the lung at 150 min for AMO/PF6 group.The T/L of AMO/PF6 group was the highest compared with AMO/LIP group and AMO group,and the difference was statistically significant(T/L:1.716±0.002 vs.1.352±0.018,P < 0.0001).In vitro imaging of animal tissues showed that the distribution of fluorescence signal in other tissues was consistent with that in vivo imaging except heart and stomach.In the frozen section,the distribution results were consistent with that of in vitro tissue imaging.Conclusion Fluorescently labeled novel AMO/PF6 nanoprobes had the advantages of simple preparation,low cytotoxicity,high transfection efficiency and excellent anti-sense imaging performance of targeted mirna-21 in A549 lung adenocarcinoma xenograft. |
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