A multiple-functionalized targeting delivery system was prepared by self-assembly for efficient delivery of Cas9/sgRNA plasmids to targeted tumor cell nuclei. The Cas9/sgRNA plasmids were compacted by protamine in the presence of calcium ions to form nanosized cores, which were further decorated by peptide and aptamer conjugated alginate derivatives. With the help of the nuclear location signal peptide and AS1411 aptamer with specific affinity for nucleolin in the tumor cell membrane and nuclei, the delivery vector can specifically deliver the plasmid to the nuclei of tumorous cells for knocking out the protein tyrosine kinase 2 (PTK2) gene to down-regulate focal adhesion kinase (FAK). The tumor cell apoptosis induced by genome editing is mitochondrial-dependent. In addition, FAK knockout results in negative regulation on the PI3K/AKT signaling pathway. Meanwhile, favorable modulation on various proteins involved in tumor progression can be realized by genome editing. The enhanced E-cadherin and decreased MMPs, vimentin, and VEGF imply the desirable effects of genome editing on suppression of tumor development. Wound healing and transwell assays confirm that the genome editing system can suppress tumor invasion and metastasis in edited cells efficiently. The investigation provides a facile and effective strategy to fabricate multiple-functionalized delivery vectors for genome editing.
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