AlphavBeta3 and AlphavBeta5 integrins are attractive target structures for cancer therapy as they are upregulated in tumor 27 and tumor associated host cells and play a pivotal role for tumor growth and metastasis. Gene vectors such as 28 polyplex micelles consisting of thiolated PEG-block-poly(lysine) copolymers complexed with plasmid DNA 29 can be targeted to these specific integrins by equipment with a cyclic RGD peptide. In this study, we analyzed 30 the effect of the RGD ligand on micelle endocytosis by comparing fluorescently labeled, targeted and 31 untargeted micelles in live-cell imaging experiments with highly sensitive fluorescence microscopy and flow 32 cytometry. Two micelle types with 12 kDa (PEG12) and 17 kDa (PEG17) PEG shell layers were examined to 33 evaluate the influence of surface shielding on the internalization characteristics. Our results reveal three 34 major effects: First, the RGD ligand accelerates the internalization of micelles into integrin expressing HeLa 35 cells without changing the uptake pathway of the micelles. Both targeted as well as untargeted micelles are 36 predominantly internalized via clathrin mediated endocytosis. Second, the PEG shielding of micelles has an 37 important effect on their targeting specificity. At high PEG shielding selective endocytosis of integrin targeted 38 micelles occurs, whereas at low PEG shielding targeted and untargeted micelles show comparable 39 internalization. In addition, PEG17 RGD(+) micelles induce the highest reporter gene expression. Third, 40 our data demonstrate a clear influence of the applied micelle dose on the internalization of integrin targeted 41 micelles.We propose that PEG17 shielded micelles equipped with a cyclic RGD ligand are the favored system 42 of choice for clinical therapy as they exhibit higher transgene expression, a higher specificity for integrin- 43 dependent endocytosis compared to PEG12 shielded micelles, and are functional at low doses as well. 44