Cation exchange protocols to radiolabel aqueous stabilized ZnS, ZnSe and CuFeS2 nanocrystals with 64Cu for dual radio- and photo-thermal therapy

In metal chalcogenide nanocrystals (NCs), the cations can be partially or fully replaced with other cations through the so-called cation exchange (CE) reactions. Here, we took advantage of these CE reactions to replace the cations on different chalcogenides NCs with 64Cu ions in order to radiolabel them. With respect to other approaches reported in the literature, our CE protocol is easily transferable to the clinic. It requires indeed one single step, in which the water-soluble NCs are mixed with a 64Cu copper(II) chloride solution of high specific activity, in the presence of vitamin C used as a reducing agent for Cu(II) to Cu(I)). Given the quantitative replacement of the cations of the NCs with 64Cu(I), a high radiochemical yield up to 90-95% can be reached. Provided that there is no free 64Cu, no purification step is needed, making the protocol straightforward. At the same time, the amount of NCs required for the exchange is so low (in the range of μg) that the dose of NCs shows no intrinsic cytotoxicity. This protocol works on different types of metal chalcogenide NCs. In ZnSe and ZnS NCs, the Zn(II) ions are exchanged with 64Cu (I) ions, and in CuFeS2 NCs the Fe(III) ions are exchanged with 64Cu(I). To ensure the stability of the NCs during and after the CE reaction, a multi-anchoring coating procedure based on PEG, cysteamine and poly-maleic anhydride was proven to be more efficient than the use of monothiol PEG ligands. With our approach we managed to achieve an unprecedented high specific activity, i.e. the amount of 64Cu radionuclide loaded per NC dose, to dispatch remarkable ionizing effects. Indeed, by exploiting a volumetric cations exchange, our strategy enables to concentrate a large dose of 64Cu (18.5 MBq) in a small NC dose (0.4 μg), reaching a specific activity of about 50 TBq/g. Remarkably, for CuFeS2 NCs even after the CE, the radiolabeled 64Cu:CuFeS2 NCs still show the characteristic dielectric resonance that enables the generation of heat under laser exposure for clinical use (1 W/cm2). The synergic toxicity of photo-ablation and 64Cu radiation exposure is here demonstrated in an in vitro study on glioblastoma and epidermoid carcinoma tumor cells.