Abstract
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Objectives: 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 99mTc for cellular delivery and in vivo imaging.
Methods: 99mTc radiolabeled miR-155 targeted AMO with partial 2’-OMe and PS chemical modification was prepared via the conjugation to 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 with cationic lipids and DSPE-PEG2000 could fuse PLGA to form AMO-phospholipid enveloped nanoparticle (AMO-PCNP), which delivered AMO into cytoplasm in vitro and into tumor in vivo. Further, this radiolabeled AMO-PCNP was confirmed by gel electrophoresis and evaluated for its serum stability, 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-PCNP.
Results: The labeled AMO-PCNP showed good conjugation between AMO and PCNP at the ratio more than 1:3 by gel electrophoresis. It also kept highly stable in human serum. Western blotting results showed that the unconjugated and conjugated AMOs 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-PCNP was significantly higher than that of unconjugated AMO. Furthermore, fluorescent protein (FAM) labeled AMO 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-PCNP and negative control probe in HeLa tumor xenograft models, which suggested the specific binding and effective delivery ability of 99mTc labeled AMO-PCNP in vivo.
Conclusion: This study supports the effectiveness and efficiency of cellular and in vivo delivery of 99mTc radiolabeled AMO-PCNP in cervical tumor and suggests a prospective candidate for future tumor imaging. Research Support: This study was supported by grants from the National Natural Science Foundation of China (NSFC 81101065), National Major Scientific Equipment Special Fund (2011YQ03011409), the Higher Education Doctoral Program of China Research Fund for New Teacher (20110001120043) and Beijing Capital Special Development Application Program (Z141107002514159).