Delivery of linear gene-editing systems by cell-penetrating magnetite vehicles: synthesis, characterization and preliminary in vitro testing

One of the main challenges in gene therapy is the transport of genetic material into target cells. This is mainly due to the need for overcoming several obstacles like rapid genetic material degradation by the physiological environment, low endosomal escape, and limited cell uptake. A meaningful way to increase the efficacy of genetic material delivery is to incorporate magnetite nanoparticles to transport biomolecules with high biocompatibility and relative ease of handling. Moreover, magnetite offers the possibility of controlled fate by magnetic fields and excretion as ferritin. This study aims to develop a nanostructured platform for the immobilization and intracellular release of nucleic acids for gene therapy applications. The system also co-immobilized the potent cell-penetrating protein OmpA (outer membrane protein A). The delivery of the conjugated material was first transiently tested in vitro in the presence of reducing agents via spectrofluorimetry. This was achieved by the presence of a reducible linker in the nanoplatform where the fluorophore rhodamine B was conjugated for proof-of-concept purposes. Based on the in vitro results, we decided to deliver this to neuroblastoma and Vero cells to confirm an endosomal escape of about 85% as calculated by colocalization. Future experiments will be focused on the hybridization of a gene sequence for the expression of the fluorescent protein mCherry. The obtained nanobioconjugate will also be delivered to cells to evaluate transfection efficiencies.