Purpose

The project aims to explore the potential of self-assembled, nanoparticulate, biocompatible, polysaccharide polymer gels containing Gd³⁺ ions to act as a high relaxivity contrast agent platform.

Background

At the magnetic field strengths employed in current clinical MRI machines the molecular tumbling rate of contrast agents is generally the limiting factor in their efficiency (relaxivity). Strategies to deliver high loads of Gd³⁺ ions with slow local tumbling have concentrated on appending Gd³⁺ chelates to macromolecular systems. Generally these approaches provide only modest gains in relaxivity and would not be practical commercially due to scaling or safety considerations. An area that has received comparatively less attention until very recently is the incorporation of contrast agents into nano-sized hydrogel (nanogel) systems.^1-4 Hydrogels are three-dimensional hydrophilic polymer networks that can absorb and retain large quantities of water, saline or physiological solutions. Using a nanogel approach has several advantages; nanogels are stable and have well defined physical and chemical characteristics in aqueous media, they can be highly loaded with molecules of interest, provide fast water diffusion and highly accessible but immobilised metal centres. Because of these favourable properties nanogel systems are seeing increased attention in the fields of drug delivery and theranostics.

Results and Discussion

Several naturally occurring, biocompatible polysaccharide polymers spontaneously undergo a gelling process in the presence of metal ions (ionotropic gelation). Alginates are the most widely used and due to their low-toxicity, low cost and relative ease of functionalisation they have received intense investigation for use in biomedical applications such as wound healing, tissue engineering and drug delivery.⁵ Nanogels containing Gd³⁺ ions can be prepared simply by ionotropic gelation of sodium alginate with Gd³⁺ solutions via nebulisation ⁶ or high dilution ⁷ methods in a similar manner to Ca²⁺ or Zn²⁺ crosslinked particles (Fig. 1). These nanogels were found to be stable in millipore water and to have a size of 100-200 nm which was confirmed by SEM and Dynamic Light Scattering (DLS). Such a size is seen as the ideal for in vivo applications.⁸ These Gd³⁺-crosslinked particles show high R₁ (and R₂) relaxivities and have the potential to provide MRI contrast with an R₁ relaxivity at 1 T of ≈ 30 mM^-1 s^-1.However, crosslinked alginate gels are relatively mobile at a molecular scale ⁹ and these ionically crosslinked particles show poor stability in the presence of high concentrations of metal ions such as Na⁺ in saline solution. In order to provide increased structural stability the particles can be further covalently crosslinked by a suitable cross-linking agent e.g. epichlorohydrin. In an idealised, completely crosslinked, situation the gadolinium ions can now be thought of as being chelated by macrocyclic ligand systems providing increased thermodynamic and kinetic stability to transmetallation. The covalent crosslinking also provides a more rigid local environment for the Gd³⁺ ion and leads to a dramatic increase in R₁ relaxivity to ≈ 60 mM^-1 s^-1 at 1 T. The stability of these crosslinked nanogel particles towards metal leaching was investigated by measuring changes in their relaxivity after introduction into solutions of NaCl, ZnCl₂ or PBS buffer (Fig 4). The results indicate an immediate dramatic decrease in relaxivity corresponding to a rapid swelling of the particles in high ionic strength media, supporting experiments show that this relaxivity change is not due to immediate loss of large quantities of Gd³⁺ from the particles. In NaCl solution there is slow transmetallation before equilibrium is reached. Addition of Zn²⁺ ions and HPO₄^2- ions show similar behaviour but now the reduction in relaxivity due to initial swelling is greater. Precipitation of insoluble GdPO₄ occurs over a period of days/weeks in the presence of HPO₄^2-. Optimisation of the physical/rheological properties of the nanogel particles may reduce these processes to a viable level.

Conclusions

We have shown a novel, promising, simple nanogel approach towards achieving extremely high relaxivity contrast agents with a 5-10 fold efficiency increase over commercially available agents. This nanogel approach has further advantages in that other payloads can be incorporated into the particles leading to possible multi-modal imaging or theranostic applications. The responsive and adaptive nature of nanogels also lends itself readily to incorporation of smart functionality. Efforts to optimise the system to provide the highest relaxivity and stability properties by choice of polysaccharide, cross-linking agent and reaction conditions are underway.

Acknowledgements

The authors wish to thank the Daphne Jackson Trust for a fellowship to Brendan Garrett as well as the University of York and the Royal Society of Chemistry for funding.