Abstract
A nano-gene vector PEI-P123-R13 was synthesized by cross-linking low molecular weight PEI with P123 and further coupling bifunctional peptide R13 to the polymer for targeting tumor and increasing cellular uptake. The binding assessment of R13 toαvβ3 positive cells was performed by HRP labeling. The internalization pathways of P123-PEI-R13/DNA complexes were investigated based on the effect of specific endocytic inhibitors on transfection efficiency. The mechanism of intracellular trafficking was investigated based on the effect of endosome-lysosome acidification inhibitors, cytoskeleton, and dynein inhibitors on transfection efficiency. The results indicated that the bifunctional peptide R13 had the ability of binding toαvβ3 positive cellsin vitro. The modification of P123-PEI-R13 with R13 made it display new property of internalization. P123-PEI-R13/DNA complexes were conducted simultaneously via clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and possible energy-independent route. After internalization, P123-PEI-R13/DNA complexes could escape from the endosome-lysosome system because of its acidification and further took microtubule as the track and dynein as the dynamic source to be transported toward the microtubule (+) end, to wit nucleus, under the action of microfilament, and with the aid of intermediate filament.
Highlights
Cancer is the main reason of death worldwide, while gene therapy is the most potential way to eradicate cancer
The zeta potential of P123-PEIR13/DNA complexes was around 5–40 mV
The results demonstrated that clathrin-mediated endocytosis was one of the efficient approaches for P123-PEI-R13/DNA complexes to pass into cells
Summary
Cancer is the main reason of death worldwide, while gene therapy is the most potential way to eradicate cancer. A high efficiency and low toxicity gene vector plays a key role in gene therapy. Especially polyethyleneimine (PEI), have recently attracted remarkable interests. A targeting vector P123-PEI-R13 was once successfully synthesized. In order to improve cell selection and increase cellular uptake, we coupled αvβ3-targeting peptide RGDC with the cell-penetrating peptide TAT to obtain a bifunctional peptide RGDCTAT (named R13) and used R13 to modify the PEI derivate P123-PEI. The purpose of this study was to solve the efficiency-versus-cytotoxicity and tumor-targeting problems of PEI as a nonviral gene delivery vector. The new nonviral gene vector P123-PEI-R13 could reduce cytotoxicity of PEI under the circumstance that PEI showed high transfection efficiency, increase the cell selection, and improve the curative effect of gene therapy. The binding affinity, cellular uptake, and subsequent intracellular trafficking of this vector should be clarified
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