Functionalized nanodiamonds – toward innovative antitumor approaches

D-amino acid oxidase (DAAO) represents a promising and well-characterized enzyme for the ‘enzyme-activated prodrug therapy’, one of the newest frontiers for anticancer treatment. This flavoprotein catalyzes the deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS) able to favor cells death. In this preliminary work, promising drug delivery systems based on nanodiamonds (NDs) were designed to target DAAO specifically to the tumor site following possible future injection in the blood stream. NDs were functionalized with polyethylene glycol (PEG), hyaluronic acid (HA) and poly(glycerol monomethacrylate) (PGMA), to reduce aggregation and improve biocompatibility. Different conjugates were obtained by DAAO adsorption on the NDs surface. The interaction with human serum proteins was evaluated in vitro, and the formation of the protein corona (PC) was investigated via nLC-MS/MS, aiming to characterize both soft and hard corona. Because the PC provides a biological identity to the conjugates and influences their fate in vivo, this study may contribute to investigate preliminary in vitro properties of functionalized nanoparticles, conjugated with well-characterized enzyme. The cytotoxicity and the oxidative stress of the designed conjugates, induced by hydrogen peroxide, were tested on two different tumor and two different healthy cell lines, as controls. The contribution of the PC to both cytotoxicity and oxidative stress was also evaluated. Among all investigated NDs-DAAO conjugates, PEG-NDs-DAAO seemed to exhibit promising antitumor characteristics: a biocompatible PC may contribute to potentially prolong blood circulation time and the increased cytotoxicity against human breast cancer cells (SKBR3, viability of 34%) could be probably favored by interaction of PEG-NDs-DAAO-PC with tumor cells. The presence of specific proteins, like serotransferrin and Inter-alpha-trypsin inhibitor heavy chain H2, could potentially play a crucial role in cell membrane adhesion.