The D-amino acid oxidase-carbon nanotubes: evaluation of cytotoxicity and biocompatibility of a potential anticancer nanosystem

The ‘enzyme prodrug therapy’ represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric favoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing fve aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efcacy under low O2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O2 concentration. In order to evaluate the nanocarriers’ biocompatibility, PEG-MWCNTsDAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has infuenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic efect controlled by the substrate addition, against diferent tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug’s biodistribution

Unveiling the Bio-corona Fingerprinting of Potential Anticancer Carbon Nanotubes Coupled with D-Amino Acid Oxidase

The oxidation therapy, based on the controlled production of Reactive Oxygen Species directly into the tumor site, was introduced as alternative antitumor approach. For this purpose, d-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, an enzyme able to efficiently catalyze the production of hydrogen peroxide from d-amino acids, was adsorbed onto multi-walled carbon nanotubes (MWCNTs), previously functionalized with polylactic-co-glycolic acid (PLGA) or polyethylene glycol (PEG) at different degrees to reduce their toxicity, to be targeted directly into the tumor. In vitro activity and cytotoxicity assays demonstrated that DAAO-functionalized nanotubes (f-MWCNTs) produced H2O2 and induced toxic effects to selected tumor cell lines. After incubation in human plasma, the protein corona was investigated by SDS-PAGE and mass spectrometry analysis. The enzyme nanocarriers generally seemed to favor their biocompatibility, promoting the interaction with dysopsonins. Despite this, PLGA or high degree of PEGylation promoted the adsorption of immunoglobulins with a possible activation of immune response and this effect was probably due to PLGA hydrophobicity and dimensions and to the production of specific antibodies against PEG. In conclusion, the PEGylated MWCNTs at low degree seemed the most biocompatible nanocarrier for adsorbed DAAO, preserving its anticancer activity and forming a bio-corona able to reduce both defensive responses and blood clearance

Hydrophobin-Coated Solid Fluorinated Nanoparticles for 19F-MRI

Funding Information: N.A., V.D., P.M., and F.B.B. are thankful to the NEWMED project, ID: 1175999 (funded by Regione Lombardia POR FESR 2014 2020). F.B.B. and P.M. are also thankful to the project NiFTy funded by MIUR (PRIN2017, no. 2017MYBTXC). C.C. and F.B.B. are also thankful to the P2RY12 project, ID: GR‐2016‐02361325 (funded by the Italian Ministry of Health). The authors acknowledge the provision of facilities and technical support by Aalto University at OtaNano—Nanomicroscopy Center (Aalto‐NMC). Prof. Pompea Del Vecchio is acknowledged for micro‐DSC measurements on HFBII‐FNP dispersions. Publisher Copyright: © 2021 The Authors. Advanced Materials Interfaces published by Wiley-VCH GmbHIn recent years, fluorine-magnetic resonance imaging (19F-MRI) has emerged as a promising diagnostic technique, complementary to traditional proton magnetic resonance imaging (1H-MRI) and easily translatable for clinical use, providing in-depth in vivo quantification without the use of radioactive agents. This creates a need for the development of appropriate delivery systems for highly omniphobic fluorinated probes. The use of the film-forming protein hydrophobin (HFBII) represents a sustainable and simple method to invert the philicity of fluorinated surfaces. Here, the ability of HFBII to form a rigid protein monolayer on superfluorinated coatings rendering them hydrophilic is shown, a property that is also retained in biological environment. This approach is then translated to directly disperse a solid superfluorinated 19F-MRI probe, PERFECTA, in aqueous solution through the formation of core-shell hydrophobin stabilized PERFECTA nanoparticles (NPs). The obtained NPs are fully characterized in terms of morphology, magnetic properties, colloidal stability, protein corona formation, cellular viability, and imaging performance.Peer reviewe