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Due to low ability to pass through the cell membrane and lack of proof in target delivery in vivo , anti-microRNA oligonucleotide (AMO) has poor target ability to microRNA in the cytoplasm, which results in low AMO targeting efficiency and limits AMO success in vivo application. In this study, AMO-phospholipids conjugates were developed to provide the characteristics of the two phospholipids to self-assemble via hydrophilic AMO and hydrophobic phospholipid tails to overcome the AMO backbone structures and enhance the AMO delivery efficiency. This AMO-phospholipids enveloped nanoparticles was radiolabeled with technetium-99 m (99mTc) for cellular delivery and in vivo imaging. 99mTc-radiolabeled miR-155 targeted AMO with partial 2′-O-methyl (2′-OMe) and phosphorothioate (PS) chemical modification was prepared via the conjugation to N-hydroxysuccinimide derivative of S-acetylmercaptoacetyltriglycine (NHS-MAG3). This radiolabeled AMO is chemically conjugated with phospholipids to form AMO-phospholipid. The AMO-phospholipids served as a component of nanoparticles to embed the hydrophobic part. AMO-phospholipids fused with polycarboxybetaine (PCB) based distearoyl phosphoethanolamine-polycarboxybetaine (DSPE-PCB) modified cationic lipids to form AMO-phospholipid-coated nanoparticles (AMO-NP), which delivered AMO into cytoplasm in vitro and into tumor in vivo. Further, this radiolabeled AMO-NP was confirmed by gel electrophoresis and evaluated for its serum stability, cytotoxicity, inhibitory ability and cellular uptake in HeLa cells, as well as the fluorescent distribution in vitro. Moreover, its distribution and in vivo imaging were performed HeLa tumor-bearing mice after the injection of radiolabeled AMO-NP. The gel electrophoresis of 99mTc labeled AMO-NP showed good conjugation between AMO and 99mTc. It also kept highly stable in human serum. Western blotting results showed that AMO-NP could up-regulate the expression of C/EBPβ protein, one of target proteins of miR-155 in HeLa cells, proving the ability in the specific binding and inhibition. The cellular uptake of 99mTc labeled AMO-NP was significantly higher than that of bare AMO. Furthermore, FAM labeled AMO-NP displayed its ability of specific and effective distribution in tumor cells and a good stability in cellular level. After the system administration, the distribution and in vivo imaging results revealed the significant difference between 99mTc labeled AMO-NP and negative control probe in HeLa tumor xenograft models, which suggested the specific binding and effective delivery ability of 99Tc labeled AMO-NP in vivo. This study supports the effectiveness and efficiency of cellular and in vivo delivery of 99mTc radiolabeled AMO-NP in cervical tumor and suggests a prospective candidate for future tumor imaging. [Display omitted] [ABSTRACT FROM AUTHOR]

Aptamers are small molecule DNA or RNA fragments that fold into well-defined 3D structures with high specificity and affinity towards target molecules, therefore emerging as a new class of probes for radiopharmaceutical applications. In this study, a technetium‐99m (99mTc) radiolabeled nucleolin-targeted aptamer AS1411 was developed for identifying glioma in murine models. A six-carbon amine group was linked at the 5′ end of AS1411 by DNA synthesis. Following the conjugation to NHS-MAG 3 , AS1411 was radiolabeled with 99mTc. Radiochemical purity and stability were evaluated by radio-TLC and gel electrophoresis. Cellular uptake and MTT assays were carried out in the nucleon-overexpressed U87 cell line to assess the biological ability of AS1411 after conjugating with FITC or radiolabeling with 99mTc. In vivo imaging of 99mTc-AS1411 was performed in glioma tumor xenografts and 99mTc and a 99mTc-labeled non-specific negative control (NC) nucleotide were used as control groups. Biodistribution studies were performed at the terminal time point of SPECT imaging. AS1411 was successfully labeled with 99mTc with the labeling yields of 81.5% ± 3.6% (n = 5) and was verified by gel electrophoresis and gel imaging. 99mTc-AS1411 was proved to be stable in fresh human serum at room temperature for 12 h. In U87 cells, 99mTc-AS1411 showed significantly higher cellular uptake than 99mTc-NC and 99mTc (9.4% ± 2.1% vs 1.9% ± 0.4% and 0.9% ± 0.1%, P < 0.01, n = 3). FITC-conjugated AS1411 demonstrated more fluorescent signals in U87 cells than NC. In addition, 99mTc-AS1411 displayed similar cellular inhibitory ability with AS1411, which was measured by MTT assay. In U87 tumor-bearing mice, 99mTc-AS1411 demonstrated high radioactive accumulation, whereas 99mTc-NC and 99mTc only showed weak tumor uptake, suggesting the specific uptake of 99mTc-AS1411 in nucleolin-positive U87 tumors in vivo. Biodistribution results further verified the imaging results. The tumor uptake of 99mTc-AS1411 was significantly higher than that of 99mTc-NC (5.81 ± 1.55%ID/g vs 0.51 ± 0.16%ID/g, n = 4, P < 0.01). 99mTc-AS1411 can be successfully prepared via the conjugation of NHS-MAG 3 and be considered a promising imaging agent capable of identifying nucleolin-positive glioma tumors. [Display omitted] [ABSTRACT FROM AUTHOR]