Nanosystems for Molecular Imaging

The main research line is focused on the development of multimodal diagnostic procedures relying on the use of structure based designed nanosystems, properly planned to quantify the expression of molecular targets, overexpressed in tumoral and/or viral pathologies: During the COVID-19 emergency, we developed a project for a diagnostic test based on a turbidimetric method. The overall idea involves the development of a simple and low cost test capable of providing rapid diagnostic results for a large number of subjects. The objective is to design liposomal systems that display peptides on their surface, which effectively interact with the RBD domain of the Spike protein. Subsequently, the formation of a liposome/virus-like network based on the peptide/RBD interaction was confirmed, resulting in the turbidity of the solution (Figure 1).

This turbidity measurement serves as an indication of the presence of the virus in the sample under examination. Peptides targeting the RBD domain were designed, synthesized, and incorporated into the membrane of appropriately functionalized liposomes. Furthermore, the nanomolar affinity of the isolated peptides and the liposomes conjugated with the Spike RBD domain was confirmed using BLI. By utilizing extracellular vesicles (EVs) that expose a portion of the Spike protein, the necessary conditions for the formation of an aggregate based on liposome/RBD recognition were determined using DLS. 
We are developing a multi-parametric in vivo diagnostic approach (MRI/OI) to evaluate the correlation between the expression level of hCA IX, hCA XII, and pH deregulation in a xenografed breast cancer murine model. Despite significant advances in therapy, breast cancer remains the second leading cause of cancer death in women. This indicates the need for a better understanding of this complex disease. In recent years, the study of H+ dynamics in cancer has led to a new paradigm known as the pH-centric anticancer paradigm. This metabolic reprogramming involves the intracellular alkalization of cancer cells and, consequently, extracellular and intratumoral microenvironmental acidosis. Among many others, two key players in pH regulation of cancer cells are the transmembrane isoforms of carbonic anhydrases, hCA IX and hCA XII. These proteins are known to be overexpressed in many common tumors and play a critical role in hypoxia-associated tumor acidosis. The extracellular part of CAIX contains a catalytic CA domain and a region with high sequence identity to the keratan sulfate attachment domain of a large proteoglycan called aggrecan, named the PG domain (Figure 2).

Unlike hCA IX, hCA XII does not contain the PG domain. hCA IX and hCA XII have been shown to contribute to the growth and survival of tumor cells, and their expression is correlated with metastasis and resistance to therapies. Recent studies have demonstrated that although hCA IX and hCA XII have similar catalytic activities, they exhibit distinct and non-overlapping expression patterns. High expression of hCA XII is associated with better survival statistics, while hCA IX expression is associated with a more aggressive tumor phenotype and poor prognosis. However, most of this information has been obtained through ex-vivo indirect measurements, such as immunohistochemical analyses of tumor biopsies. Therefore, we are developing an in vivo molecular imaging approach using agents that selectively target hCA IX and hCA XII, which are overexpressed at sites of hypoxia. So far, there have been no Magnetic Resonance Imaging (MRI) studies on tumor imaging that exploit CAIX as a target. MRI is a technique that provides superior soft tissue contrast and excellent spatial resolution. However, the inherent sensitivity of MRI is relatively low, and the ability to visualize a molecular marker in vivo depends on the target concentration and/or the efficacy and amount of contrast agent accumulated at the site. To enhance the concentration of the contrast agent delivered to the target, the most common strategy involves the use of paramagnetic liposomes. These liposomes can transport a large amount of contrast agents per vesicle, amplifying the effect of the molecular recognition event. Their structure allows for the encapsulation of active components in the aqueous core and the incorporation of multifunctionalized phospholipids in the membrane, making them suitable for theranostics and image-guided drug delivery, including MRI imaging. We are developing a Gd-based MRI nanoprobe that carries a CAIX PG domain targeting vector on the surface and a quenched T1-contrast agent in the interior space. This allows for the specific delivery of a cargo of MRI probes to the receptor site and the tracking of its intracellular fate. As a recognition peptide for the target, we modified some sequences developed from the crystallographic structure between the PG of CAIX and one of its antibodies. The nano-system thus prepared resulted in specific in vitro MRI-labeling of breast cancer cells, up to 80 times more effective than that obtained using a scramble sequence. Interestingly, this huge specificity emerges in the MRI signal only 8 h after the end of incubation, when the probe was likely entirely internalized and has released the paramagnetic molecule into the cells (Figure 3).

This could be ascribed to the process of quenching, consisting in silencing the probe relaxing ability on bulk water signal as long as it is entrapped in the inner core of a nanovesicle at high concentration. These T1-quenched nanosystems are designed to recover the signal enhancement ability following the release of vesicle contents in response to biological events. The strategy of our MRI procedure is based on incapsulating a high concentration of contrast agent in the liposome that is quite silent in the condition of our MRI experiment, producing a high MRI signal only after the release of the contrast agent (de-quenching). In collaboration with Prof. Delli Castelli’s group at MBC, we have successfully translated the procedure in vivo using three groups of murine models with breast cancer: one group treated with the liposomes functionalized with the specific peptide, another group treated with a scramble peptide, and a control group without any peptide treatment. Consistent with previous experiments, the group treated with the specific peptide-functionalized liposomes showed a significant increase in signal enhancement. The peak of T1 signal enhancement in the tumor was observed at 8 hours after injection, indicating the internalization of liposomes into cells through PG-CAIX binding and subsequent release of the contrast media. The developed probe in this study has the ability to generate a high signal enhancement in response to the interaction between liposomes and CAIX, as well as the degradation of the vesicles. This enables sensitive detection that can be monitored in vivo through MRI.