Figure 6.

Model for delivery of shRNA (adapted from [18]). This illustration depicts cellular uptake of plasmid Tf-PEG-PEI nanoparticles and the mechanism of action of shRNA. First, a pSico vector containing a U6 promoter-loxP-CMV-GFP-STOP signal-loxP-CD44vshRNA (gene of interest) is made. Second, an expression vector with the Cre-recombinase gene controlled by the tissue specific promoter is created. Third, the two vectors are packaged in transferrin (Tf) coated-PEG-PEI nanoparticles that bind with Tf-receptors (Tf-R) present at high levels in the targeted tumor cells. Delivery of the vectors in normal and malignant cells from the targeted tissue results in deletion of the Stop signal and transcription of Cre-recombinase driven by the tissue specific promoter. The target gene (CD44vshRNA) is then unlocked and transcribed through the strong U6 promoter for high expression. The normal tissue cells are not affected because they do not make the targeted CD44 variant. Tf-PEG-PEI nanoparticle coated plasmids (pSico-CD44v6shRNA/pFabpl-Cre) circulating in blood accumulate at tumor regions enhanced by the EPR effect. Endocytosis mediated by ligand-receptor interactions occurs because the nanoparticles are coated with the Tf-ligand for the Tf-R receptor on the tumor cell surface.