Andrea Cantelli

Fluorescence, and more in general, photoluminescence (PL), presents important advantages for imaging with respect to other diagnostic techniques. In particular, detection methodologies exploiting fluorescence imaging are fast and versatile; make use of low-cost and simple instrumentations; and are taking advantage of newly developed powerful, low-cost, light-based electronic devices, such as light sources and cameras, used in huge market applications, such as civil illumination, computers, and cellular phones. Besides the aforementioned simplicity, fluorescence imaging offers a spatial and temporal resolution that can hardly be achieved with alternative methods. However, the two main limitations of fluorescence imaging for bio-application are still (i) the biological tissue transparency and autofluorescence and (ii) the biocompatibility of the contrast agents. Luminescent gold nanoclusters (AuNCs), if properly designed, combine high biocompatibility with PL in the near-infrared region (NIR), where the biological tissues exhibit higher transparency and negligible autofluorescence. However, the stabilization of these AuNCs requires the use of specific ligands that also affect their PL properties. The nature of the ligand plays a fundamental role in the development and sequential application of PL AuNCs as probes for bioimaging. Considering the importance of this, in this review, the most relevant and recent papers on AuNCs-based bioimaging are presented and discussed highlighting the different functionalities achieved by increasing the complexity of the ligand structure.

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C₆₀@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C₆₀ molecules in a physiological environment, (ii) HSA, upon C₆₀ binding, maintains its biological identity and biocompatibility, (iii) the C₆₀@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C₆₀@HSA complex in HeLa cells.

The high hydrophobicity of fullerenes and the resulting formation of aggregates in aqueous solutions hamper the possibility of their exploitation in many technological applications. Noncovalent bioconjugation of fullerenes with proteins is an emerging approach for their dispersion in aqueous media. Contrary to covalent functionalization, bioconjugation preserves the physicochemical properties of the carbon nanostructure. The unique photophysical and photochemical properties of fullerenes are then fully accessible for applications in nanomedicine, sensoristic, biocatalysis and materials science fields. However, proteins are not universal carriers. Their stability depends on the biological conditions for which they have evolved. Here we present two model systems based on pepsin and trypsin. These proteins have opposite net charge at physiological pH. They recognize and disperse C₆₀ in water. UV-Vis spectroscopy, zeta-potential and atomic force microscopy analysis demonstrates that the hybrids are well dispersed and stable in a wide range of pH’s and ionic strengths. A previously validated modelling approach identifies the protein-binding pocket involved in the interaction with C₆₀. Computational predictions, combined with experimental investigations, provide powerful tools to design tailor-made C₆₀@proteins bioconjugates for specific applications.