The use of antisense oligonucleotide molecular beacons, able to generate a fluorescent signal when they hybridize with their target mRNA, represents an innovative strategy in cancer. This approach is able to conjugate the ability of sensing specific mRNA with the pharmacological silencing activity, preventing the overexpression of proteins associated to cancer development. In this context, this strategy minimizes the non-specific toxicity addressing the therapy mainly towards the tumor cells by using effective delivery systems. The aim of this work was to investigate the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to act as vehicle of an oligonucleotide molecular beacon (MB) targeting survivin mRNA in A549 human lung adenocarcinoma epithelial cells. Furthermore, this paper focuses the attention on the need for having an appropriate healthy control in in-vitro experiments. In particular, the survivin-MB was firstly characterized in solution in order to verify its functionality and then the PMMA-NPs ability to promote the MB internalization was verified in A549 cells by confocal microscopy. Confluent Human Dermal Fibroblasts from adult (HDFa) were used as healthy control. The results showed that PMMA-NPs promote the survivin-MB cellular up-take and that the use of 10 μg/mL PMMA-NPs as carrier for survivin-MB for 1h 30 min might be a promising strategy to reduce cancer cell proliferation avoiding detectable consequences on the healthy cells.