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Osteosarcoma (OS), the main primary malignancy of bone, is the second leading cause of cancer in children and young adults. Despite the advances in modern treatments, the 5-year survival rate is retained in 60–70%, since the conventional treatment options available are associated with relapse, chemoresistance, and development of metastases, which frequently lead to patients death. In this regard, there is an increasing need to search and develop novel and alternative therapeutic approaches. Concerning this, gene therapy appears as an innovative and promising treatment option. This therapeutic option aims to deliver genetic material, through nanosystems, to repress or replace the expression of mutated genes involved in important regulatory pathways. To attain this goal, gene therapy is decidedly dependent on the efficiency of utilized vectors, constituting such a very important parameter to take in consideration. In this work, the main goal was centered on the development and full characterization of an efficient micellar nanosystem, based on the chemical conjugation between the amphiphilic copolymer Pluronic® L64 and the cationic polymer polyethyleneimine (PEI), to deliver the therapeutic miRNA-145 into OS cells leading to inhibition of cell proliferation and migration, and ultimately inducing cell death, crafting a novel anticancer therapeutic approach to OS.