Molecular Imaging is a cutting-edge medical technique that enables the visualization and analysis of biological processes at molecular and cellular levels within living organisms. The main imaging technologies that are used to this purpose, at clinical or preclinical level, are: positron emission tomography (PET), single-photon emission computed tomography (SPECT), optical Imaging (OI), computed tomography (CT) and magnetic resonance imaging (MRI). Each of these techniques exhibits weaknesses and strengths for these applications. For instance, PET is the most sensitive among the imaging techniques but employs radiotracers and has low spatial resolution. Optical imaging is highly sensitive and minimally invasive, but it is limited in tissue penetration capability. MRI provides highly detailed anatomical information with good sensitivity, although much lower compared to PET and optical imaging. For these reasons, quite often, these techniques work in synergy taking advantage of hybrid instruments. Molecular Imaging approach holds immense promise in various fields, including oncology, neurology, cardiology, and immunology, as it provides insights into disease progression, early detection, and treatment response assessment. The combination of advanced imaging technologies with targeted molecular probes empowers scientists to unravel the intricacies of complex biological processes, leading to enhanced diagnostic accuracy and to the development of personalized therapeutic strategies. The research activity of Prof. Delli Castelli and her team belong to this broader area of Molecular Imaging. In particular, in the last years, they have contributed to the advancement of research in the field of contrast agents for Magnetic Resonance Imaging for molecular targeting and metabolic imaging applications. Within the array of magnetic resonance imaging contrast agents, this group has specialized in advancing a relatively emerging category referred to as Chemical Exchange Saturation Transfer (CEST) contrast agents. The distinctiveness of these contrast agents, as opposed to conventional gadolinium-based ones, lies in their capability to simultaneously visualize multiple entities within a single image. This unique feature allows the visualization of multiple epitopes simultaneously in molecular targeting experiments or the visualization of more than one cellular population in cell tracking applications. Such implementation cannot be achieved in MRI using conventional gadolinium-based agents since the response these molecules induce in the system is undistinguishable from one molecule to another, leading to a reduction in bulk water relaxation time. In contrast, CEST agents provide the opportunity to introduce a frequency-encoded contrast, capitalizing on the distinct chemical shifts of the molecules under observation like in magnetic resonance spectroscopy (MRS); however, CEST-MRI enhance the detection sensitivity through the indirect visualization of molecules at low concentration on the much more intense bulk water signal, resulting in an amplified response. Another distinct characteristic of this category of contrast agents, as opposed to conventional ones, is that CEST contrast can be easily modulated through tissue microenvironment parameters. Consequently, these molecules have proven to be excellent reporters of temperature, pH, redox potential, and catalytic activity. Despite these significant potentials, CEST contrast agents suffer from low sensitivity, a characteristic that the scientific community involved in their development has constantly addressed. One of the primary contribution of this research team regarding the sensitivity issue has focused on the development of nanosystems. These nanosystems, called LipoCEST have led to a remarkable increase in sensitivity by several orders of magnitude. This advancement has shifted the detection thresholds from millimolar concentrations to nanomolar concentrations, which are much more aligned with the purposes of molecular imaging. Due to the exceptional versatility of LipoCEST, these nanovesicles can be readily customized with molecular targeting vectors. Furthermore, these systems have been engineered to align themselves in a magnetic field accordingly with the sign of the magnetic susceptibility of their membrane thus altering the chemical shift (LIPO) of the mobile protons connected to these systems. This innovation facilitates the establishment of a library for multifaceted visualization (Fig. 1).
Regarding applications in metabolic imaging, the contribution of this research team has predominantly revolved around the advancement of pH-responsive probes, particularly involving paramagnetic molecules (ParaCEST agents). One of the most intriguing ParaCEST agents that has been developed is the YbHPDO3A complex. In solution, this probe exists as two isomers in slow exchange on the NMR timescale. This unique characteristic permits the visualization of two distinct signals deriving from the –OH protons of the two distinct isomers (SAP and TSAP). Fortunately, these two hydroxyl protons exhibit different CEST responses with respect to pH variations, allowing for a ratiometric approach to determine pH independently from the total probe concentration (which in vivo remains unknown). Figure 2 illustrates the pH map of the tumor microenvironment within a melanoma mouse model, obtained using the YbHPDO3A probe. The probe prove to be sensitive in the physio-pathological pH range.
In parallel with the research in the CEST field, this team has recently exploited their expertise in nanosystems to contribute to in vitro diagnostic test development. One example is reported in Fig.3 where a schematic representation of an alternative to the ELISA test (named LICIA, LipHosome Congiugated Immunoassorbent Assay) based on the use of liposomes able to change the pH in their environment following their dissembling is reported. The developed reporting systems for ligand/antiligand assays based on pH variations have been called LipHosomes. These tests have shown a strong competitive edge, particularly in cost effectiveness, when compared to the ELISA counterparts.

Future research plans