269987 Microrna-29b Delivery Via EpCAM Targeted Cationic Lipoplexes in Lung Cancer Treatment
Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. MicroRNAs are non-coding RNAs which regulate fundamental cell processes and have the potential to target multiple genes simultaneously, i.e. “one for all”. MicroRNAs have been implicated in lung cancer development by serving as tumor suppressors or oncogenes. However, miRNA-based therapeutics are faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report an EpCAM (epithelial cell adhesion molecule)-targeted cationic lipoplexes based miRNA delivery system to address some of these challenges.
Among many lung cancer related miRNAs, miR-29b, a tumor suppressor, was selected as the representative therapeutic target because it has been associated with patient survival rate and sensitivity to chemotherapy. The miR-29b delivery efficiency of EpCAM targeted lipoplexes was first evaluated in A549 non-small cell lung cancer (NSCLC) cells and compared with siPORT™ NeoFX™ transfection agent (Life Technology Inc.). The expression of mature miR-29b and its 3 target genes (CDK6, DNMT3b, MCL-1) was measured using qRT-PCR and western blotting. The A549 cell growth and sensitivity to chemotherapeutic agents were characterized 24h after miR-29b overexpression. Subsequently, both the biodistribution and cytotoxicity in vivowere evaluated in ICR mice.
Compared to NeoFX, EpCAM targeted lipoplexes delivered miR-29b in a more efficient manner with ~10.5 fold increase in mature miR-29b expression in A549 cells. Results of flow cytometry and confocal microscopy demonstrated that the higher mature miR-29b expression was led by higher cellular uptake of EpCAM targeted lipoplexes. MiR-29b delivered by EpCAM targeted lipoplexes successfully down-regulated the expression of 3 target genes, CDK6, DNMT3b, MCL-1, by 61%, 40% and 21% respectively, but only by 20%, 22% and 17% with NeoFX. miR-29b delivered by lipoplexes significantly inhibited A549 cell growth by 62.3%, compared with 18.4% by NeoFX. Overexpression of miR-29b increased the sensitivity of A549 cells to chemotherapeutic agents. The IC50 value of cisplatin for A549 cells was 7.90uM 24h after miR-29b delivery by lipoplexes, compared to 12.94 uM by NeoFX. In vivo, EpCAM targeted lipoplexes achieved ~30% accumulation in lung tissue, which was ~50 fold higher than NeoFX. Lipoplexes mainly accumulated in pneumocytes, a region for lung cancer initiation. No short term cytotoxicity was observed in vivo. Mice systemically treated by miR-29b containing lipoplexes had a 23.6-fold induction in mature miR-29b expression in the lung compared to untreated mice.
Our results have demonstrated that the novel EpCAM targeted cationic lipoplexes may have potential as carriers for miRNA based therapeutics in NSCLC treatment. In the future, further in vivo miRNA-based delivery studies will be done to evaluate long-term local and systemic toxicity and target gene effects prior to clinical application.
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