Covalent functionalization of nanodiamonds for bioapplications

In the last years, nanoscale diamonds are emerging as important materials for a variety of high performance technologies, thanks to their characteristics as the presence of a stable inert core and an alterable surface chemistry, the small primary particle size (from 4-5 nm to about 20 nm) and, in some cases, the fluorescence. Nanodiamonds (NDs) have been demonstrated to be promising tools for the delivery of biomolecules into cellular systems and to act as fluorescent label at the same time. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Nanodiamonds are characterized by a sp3 diamond core, surrounded gradually by a distorted-strained diamond layer and by an envelope of non-crystalline carbon that is a mixture of sp2 and sp3 hybridized atoms. The surface of pristine nanodiamond particles contains a complex array of functional groups as carboxylic acids, esters, ethers, lactones, amines, etc. so several chemical modifications could be introduced. Pristine detonation nanodiamonds were fully characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (pXRD), Raman, infrared (IR) and UV-visible spectroscopies. The surface of nanodiamonds was oxidized using a mixture of sulfuric and nitric acids in order to make the surface more homogeneous and to increase the number of carboxylic groups. The amidation on pristine and oxidized NDs was studied using both thionyl chloride and O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) as coupling reagents. The amine used was the tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate, so after the Boc- deprotection a further grafting of the NDs was performed with fluorescent dyes or electrochemical active moieties. On the NDs surface it was also possible to obtain a sp2 hybridized atoms’ layer by means of annealing processes, so addition reactions on double bonds, typically used for the functionalization of carbon nanotubes, were applied. The arylation with functionalized aryl diazonium salts, the 1,3-dipolar cycloaddition and the nitrene reactions were applied on annealed NDs studying both classical conditions and microwave irradiation. Moreover, oxidized NDs were functionalized with tumor homing and cell-penetrating peptides. The presence of this kind of peptides on the nanodiamond surface could be useful for the drug delivery and at the same time it can improve the dispersion stability. Tumor homing sequences and cell-penetrating peptides (CPPs) were synthesized using the solid phase peptide synthesis (SPPS) approach and they were fully characterized by reverse-phase high pressure liquid chromatography (RP-HPLC) and high resolution mass spectroscopy.