ad D-amino acid oxidase (DAAO) is an enzyme which generates reactive oxygen species (ROS) and it is believed to have potential uses as a novel therapeutic molecule if internalized by cancer cells or if they are localized close to their plasma membrane. When conjugated onto iron oxide nanoparticles (NPs), the enzyme can be magnetically directed to targeted locations with an increased efficacy. A subsequent injection of DAAO substrate D-alanine can initiate ROS production and induce apoptosis of cells surrounding the NP-DAAO complex. Here, we describe a platform for optimal bioconjugation using monodisperse g-Fe 2 O 3 NPs ($10 nm) resulting in high DAAO loading, stable NP-DAAO dispersions and more than 90% enzymatic activity recovery, which is retained using the particles in human serum. Lastly, since the NP-DAAO system is designed for cancer therapy, we proved its efficacy in killing SKOV-3, U87 and HCT-116 cancer cells.Nanobiotechnology has found widespread applications 1 in biomedicine as diagnostic imaging tools, 2,3 drug delivery systems, 4,5 vaccines 6,7 and in immunotherapy. 8 NPs can be employed to produce a great variety of bioconjugates by graing biomolecules onto a NP carrier. In oncology, for example, recent studies have shown that engineered NPs are capable of reaching specic targeted areas of the body and of releasing the therapeutic drug locally.9-12 This novel approach has the potential to increase the drug efficacy and to eliminate the side effects.
13To guide the bioconjugates through the body, one can use magnetic NPs that, aer intravenous administration, can be directed to the cancerous tissue with an external magnetic eld.14 Magnetic NPs alone are used as an effective anticancer agent during hyperthermic therapy in which NPs exposed to an alternating magnetic eld produce heat that kills all the surrounding cells.
15-19D-Amino acid oxidase from Rhodotorula gracilis (RgDAAO, EC 1.4.3.3) is a avoenzyme that utilizes D-amino acids to produce reactive oxygen species (ROS) 20 and can be used to induce apoptosis. In particular the co-administration with D-alanine as substrate, gives the corresponding a-keto acid, ammonia and H 2 O 2 . Despite the benet in using this enzyme in therapeutic applications, the main limitations are the low enzyme stability, the low efficacy in cellular internalization and the lack of specic molecular targeting. To overcome these limitations, in the past few years different attempts have been made in the development of NP-DAAO systems where the enzyme was covalently bound to the amino groups of APTES functionalized NPs. That resulted in an increased cellular internalization of the enzyme, a prolonged enzymatic activity and storage stability, and the possibility of targeting to the cancer mass with the help of an external magnetic eld. However, while the APTES functionalization represents a good amino group donor, its activation via glutaraldehyde can affect the enzyme integrity 21 leading to a loss of DAAO activity.22,23 Cappellini et al. 24 reported a higher yield...