Impact of biopolymer matrices on relaxometric properties of MRI contrast agents and their application to Nanotechnology

Magnetic Resonance Imaging (MRI) represents the first-line diagnostic imaging modality for numerous indications. It is a clinically well-established, non-invasive technique providing three dimensional whole body anatomical and functional imaging. It takes advantage of the magnetic properties of water protons present in the body and their tissue-dependent behaviour. High magnetic fields (1.5 T and above) are clinically favoured because of their higher signal-to-noise ratio, capability for MR spectroscopy, and other forms of functional MRI, high speed imaging, and high resolution imaging. Signal intensity in MRI is related to the relaxation rate of in vivo water protons and can be enhanced by the administration of a contrast agent (CA) prior to scanning. These CAs utilize paramagnetic metal ions and enhance the contrast in an MR image by positively influencing the relaxation rates of water protons in the immediate surroundings of the tissue in which they localize. Among different CAs, Gadolinium contrast medium is used in up to 30% of MRI scans and the most clinically-used MRI. However, Gadolinium (Gd), like most of the clinically-used CAs, is characterized by a relaxivity well below its theoretical limit, lacks in tissue specificity and, in addition, it causes heavy allergic effects and serious nephrotoxicity. In this framework, Port et al. have reported that the rigidification of MRI CAs, obtained through covalent or non-covalent binding to macromolecules, could be favourable to an increase in relaxivity of the metal-chelate. Later, Decuzzi et al. have proved that it is possible to modify through the geometrical confinement the magnetic properties of MRI CAs by controlling their characteristic correlation times without the chemical modification of the chelate structure. Furthermore, Courant et al. have highlighted the capability of combined hydrogels to boost the relaxivity of Gd-based CAs. Despite several experimental studies addressed in this field, a comprehensive knowledge of the mechanisms involved in the relaxation enhancement due to the entrapment of CAs in polymer-based architectures is still missing. In particular, the role played by the water at the interface between polymer chains and MRI CAs has not been fully investigated and could lead to the availability of tailored models that accurately describe these novel complex systems. In this work, we aim to demonstrate that a more in-depth knowledge about the interference between macromolecules and MRI CAs and an understanding of their physicochemical properties can significantly to impact in the design strategies of the nanostructures and, consequently, to overcome the limitations of clinically used MRI CAs, particularly linked to the low relaxivity. In this perspective, it is of primary importance to study the main phenomena involved in the formation of polymer matrices and how their properties can influence the relaxivity of MRI CAs. For this reason, we proposed a general strategy based on formation of nanostructures for boosting the efficacy of commercial Gd-based CAs by using FDA approved biopolymers, providing also tissue specificity and reduced nephrotoxicity. Indeed, we want to take advantage not only by the use of nanotechnologies for enhanced MRI but only by their capability to reach a specific target and to accumulate only in the site of interest. The implemented strategy has consisted in the control of the relaxometric properties by tuning the water dynamics, the physicochemical interactions and, therefore, the polymer conformation. Effectively, we primary investigated, in bulk, the impact of hydrogel solutions on the relaxometric properties of commercial CAs, highlighting the key role of hydrogel structural parameters (mesh size and crosslink density) in the relaxation enhancement. In this part, chemical and thermodynamic interactions involved in the complexation between biopolymers and CAs have been investigated through Isothermal Titration Calorimetry. Furthermore, characterizations of water dynamics and mobility and measurement of the relaxometric properties in hydrogel solutions containing CAs have been carried out by NMR and Time-Domain relaxometer. The main outputs were summarized in a concept called Hydrodenticity and defined as the equilibrium between the water osmotic pressure, the elastodynamic forces of the polymer chains and the hydration degree of the CA which is able to increase the relaxivity of the CA itself. Indeed, hydrogel nanostructures made of hydrophilic polymer chains held together by chemical or physical crosslinking, have the ability to swell in water, forming elastic gels that retain a large quantity of fluid in their mesh-like structures. The presence of hydrophilic polymer interfaces and the control of water behaviour in hydrogels play a fundamental role in the relaxation enhancement of the Gadolinium-based CAs by influencing the characteristic correlation times defined by the theory of Solomon and Bloembergen. Then, starting from the acquired knowledge, we moved to observe the role of Hydrodenticity in the design of biopolymer nanostructures for enhanced MRI. For the nanostructures’ synthesis, we used two different processing techniques: (1) High Pressure Homogenization; (2) Microfluidic Flow Focusing. These techniques were selected because of their ability to control process parameters enabling the tuning of the interaction between the biopolymers and the CA. Indeed, by easily adjusting concentrations, pressure of the Homogenizer and/or flow rates of the Microfluidic platform, we can modulate the crosslinking degree of the nanostructures and tune their hydrophilicity, size, shape and surface charge, impacting on the relaxometric properties. These approaches allow us to load MRI CAs into functional nanostructures and obtain nanocarries with tunable relaxometric properties. The powerful aspect and the novelty of our approach lies in the definition of Hydrodenticity and in its application to several architectures, biopolymers, lipids and mixture of them., preserving the main properties of nanoparticles for drug delivery. As future perspective, the nanostructures can also be engineered to carry more than one agent, accumulate in specific tissues or to act as probes for simultaneous diagnosis and therapy (theranostic or multimodal imaging agents), thereby facilitating targeted treatments and precision medicine.