(198ak) Nanoescapology Enabled By Surface-Engineered Magnetite: Novel Routes for Targeted Drug Delivery

Nanomedicine has become one of the most attractive avenues to promote targeted delivery and increase bioavailability of drugs that fail to spontaneously pass the cell membrane due to their high hydrophobicity and low solubility. As a result, over the past two decades an enormous body of knowledge has been developed where nanostructured materials served as the main platforms for conjugation and subsequent intracellular delivery of various pharmacological agents for the treatment of different conditions including several types of cancer, Alzheimer´s and Parkinson´s. Despite the important success both in vitro and vivo of the proposed vehicles, a major challenge is to assure that the delivered therapeutics remain active upon delivery. This is mostly due to the formation of endosomes that act as traps for the delivered molecules. To overcome this major issue, we proposed the conjugation of a transmembrane protein from E.Coli to nanostructured materials and particularly to magnetite nanoparticles, which were prepared by co-precipitation of Iron Chlorides. Immobilization was conducted after silanation, which rendered free amine groups on the nanoparticles’ surface. Protein molecules were then conjugated with the aid of glutaraldehyde to the N-terminal by forming imine bonds. As-synthesized and silanized magnetite size distribution in distilled water at 1 mg/mL was estimated via DLS. Immobilization was verified with the aid of FTIR analysis, SDS-PAGE and BCA assay. As-synthesized magnetite nanoparticles exhibited an average diameter of 120 ±62.7 nm, which was maintained even after silanation. Successful protein conjugation was determined by the absorption bands of the silanation agent and the protein. Accordingly, vibrational bands at 3000 cm^-1 and ~1600 cm^-1 were identified and correlated to the terminal -NH₂ group of the silanation reagent APTES while the protein was correlated to the vibration of the amide I band at about 1650 cm^-1. The vehicle was successfully tested in liposomes, Vero cells and Monocytes. Briefly, fluorescently-labeled proteins were immobilized, delivered and imaged with the aid of a confocal microscope. Our findings suggest that the obtained nanobioconjugate is capable of translocating the cell membrane and bypassing intracellular endosomal routes. Additionally, citotocixity assays on Vero via LDH cells suggested a rather high cell viability for nanobioconjugate concentrations below 100 ug/mL. Due to the potency of this novel vehicle, we are exploring applications in the delivery of therapeutics for the treatment of Parkinson´s disease in primary Astrocyte culture as well as anticancer compounds in a colon carcinoma cell line.