The cellular membrane constitutes an effective barrier that protects the complex, yet highly ordered, intracellular compartment of the cell. Passage of molecules across this barrier is highly regulated and highly restricted. Cell penetrating peptides (CPPs) are a class of small cationic peptides that are able to defy the rules of membrane passage and gain access to the intracellular environment. MPG is one member of this class of cell penetrating peptides. In this study, MPG was used to deliver double-stranded siRNA into human multipotent stem (hMADS) cells. Confluent and differentiated hMADS cells were transfected for 4 h with 30 nM siRNA-MPG-complex (SMC) and the controls were overlaid with free siRNA (SO) and analysed after 24, 48 and 72 h incubation post-transfection for internalization of the siRNA complex using Zeiss Axioimager. There was no noticeable adverse morphological variation between the siRNA-MPG complex transfected groups and the ‘free siRNA’ and the ‘cell only’ control groups. There was also no observed cytotoxicity associated with transfection. The differentiation of the cells into adipocyte phenotype was observed normally in all the groups. Bright fluorescence speckles were detected in the SMC transfected cells at all the 3 time points in the cytoplasm as well as in the nucleus 24 and 48 h post-transfection. The efficiency of delivery 24 h after transfection in the confluent cells was about 90%, in cells differentiating three days toward adipocytes about 80, and 50 to 60% of the cells showed internalised siRNA-MPG complex even 48 and 72 h post- transfection. This study shows that MPG efficiently mediated cytoplasmic and nuclear delivery of double-stranded siRNA into human mesenchymal stem cells under mild conditions in proliferating and in differentiation stages. These results demonstrate that MPG is a very effective and robust non-viral based transfection agent, easy to apply to non-dividing adherent cells in situ. Thus, MPG is a valuable tool for transient gene and microRNA silencing in vitro.
Key words: Cell-penetrating peptides, microRNA, MPG, siRNA, stem cells, transfection, delivery, nanoparticles.
DMEM, Dulbecco’s modified Eagle’s medium; SMC, siRNA-MPG complex; SO, siRNA only; FAM, 6-carboxy fluorescein; DAPI, 4’.6-diamidino-2-phenylindole; CPPs, cell penetrating peptides; hMADS, human multipotent stem cells; PTDs, protein transduction domains; CO, cell-only; MIP, maximum intensity projection.
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