Engineered extracellular vesicles of mesenchymal stem cells as a tool for targeted tumor suppressor miRNA delivery

Abstract

Background & Aim It is well established that stem cells (MSC) exhibit their therapeutic effect via paracrine mechanisms, including extracellular vesicles (EV). EVs interact with target cells and deliver their contents impairing their therapeutic effect. One of the potential applications of such EVs could be tumor suppressors miRNA delivery to cancer cells. However, there is an issue with a specific EV deliver to target cells. Therefore the aim of this study is to design targeted MSC EVs with specific sorted tumor suppressor miRNA inside. Methods, Results & Conclusion In order to reach the goal commercially available adipose-derived stem cell culture ASC52telo -immortalized with hTERT was selected for modification. Cells were modified with a hybrid gene that contains MSC EV surface marker and antibody binding protein by lentivirus. XMIRXpress system was used to sort tumor suppressor miRNA - 206 into modified EVs. Hybrid gene cloning was confirmed by PCR and hybrid gene expression by WB and high-resolution flow cytometry. miRNA expression and EV sorting were confirmed by qPCR. EV uptake and miRNA - 206 functionality was confirmed by flow cytometry and qPCR. While the effect of EV uptake on different cancer cells where tested by scratch and proliferation assays and qPCR on proto-oncogenes. There were developed three hybrid genes in order to increase the success rate of modification. Two of them were confirmed to expressed into MSC EVs. High-resolution flow cytometry showed that part of EVs contains a hybrid protein on the surface while qPCR showed that miRNA-206 was significantly more sorted into EVs of modified MSC cells. Next, modified EVs where stained and coupled with antibodies against breast cancer cell line MCF7 surface antigen and uptake was tested with a flow cytometer. These results showed that EV coupled with antibodies have higher uptake efficacy and target genes of tumor suppressor miRNA-206 where downregulated, resulting in decreased cell malignancy. In summary, this study shows proof of the principle that modified EV of MSC containing tumor suppressor miRNAs can be used as a potential targeted therapy strategy.