IsoThermal Calorimetry as a Probe of Interactions between Magnetic Nanoparticles, Plasma Proteins and Cells – ITC-NanoProbe

1 Background Magnetic nanoparticles are currently used in a wide variety of material science and biomedical applications. In biomedicine, they are primarily utilized as contrast agents in Magnetic Resonance Imaging. Their specific accumulation in cells or tissues permits, through the modification of the proton relaxation time, a visualization of suspected locations and organs. In addition to these properties, magnetic nanoparticles also play an important role in cell labeling, nucleic acid/protein separation, magnetically guided drug delivery and hyperthermia. In all these examples, applications require that the magnetic nanoparticles remain stable and disperse in physiological conditions and biological environments. When particles irreversibly aggregate, they lose some of their features such as their large surface-to-volume ratio and their property to 'get close' to a biological entity of interest. During the last years, the colloidal stability of magnetic nanoparticles has become a key issue in the control and design of novel nanostructures. To increase their colloidal stability, magnetic nanoparticles are often coated with polymers. Polymers have appeared as excellent candidates for many reasons. One of these reasons is that polymers can build a diffuse and protective shell around the particles, thereby increasing the overall colloidal stability. Our approach of the association between polymer and particles is based on electrostatic complexation. 10 nm magnetic nanoparticles and biocompatible polymers, such as polypeptides and polysaccharides will be synthesized to this goal. 2 Goals As a first goal, the proposed research aims to develop self-assembled functional nanostructures with controlled hierarchical morphologies, based on non-covalent binding. In this context, the particles and polymers will be the building blocks for novel nanostructures, such as core-shells, clusters, and filaments. The strategy will deal with coating, complexation, coacervates (the 3C's) which denote three possible states for nanoconstructs built up from polymers and nanoparticles. The second goal of this project is to use Isothermal Titration Calorimetry (ITC) to investigate and quantify the interactions between the magnetic nanostructures and biological entities. ITC is a thermodynamic technique for monitoring any physico-chemical reaction initiated by the addition of a binding component. It was developed to study the specific binding occurring in biological processes and yielding to the molecular organization of living matter. In this work, ITC will be complemented by investigation techniques of soft condensed matter, such as scattering (neutron, x-ray, light) and microscopy (AFM, TEM, Fluorescence). 3 Phases The first phase of the project will be to produce hybrid nanostructures using different pathways. Nanoparticles coated with a thin polymeric layer will be a major topic because their overall sizes are kept in the range 10 - 20 nm, a property of interest for biology. In this context, a crucial task will be to establish a link between the microstructure (i.e. scattering), the colloidal stability (i.e. phase behavior) and the thermodynamics (i.e. ITC). It is anticipated that high binding constants associated with exothermic reactions should guarantee a robust adsorption and a resilient coating of the organic species. In a second phase, ITC will serve to quantify the adsorption of model proteins on magnetic nanoparticles and nanostructures, such as those involved in opsonisation. Opsonisation is the process whereby plasma proteins e.g. immunoglobulins recognize micro-organisms present in the blood compartment. Our in vitro approach of opsonisation will deal with the study of the phase behavior and thermodynamics of nanostructures in solvents with increasing complexity. Bovine serum albumin (BSA), immunoglobulin (Ig) and fibrinogen proteins will be central to the study. In a third phase, experiments between nanostructures and living cells such as fibroblasts or macrophages present in the blood compartment will be attempted in the context of stealthness and cytotoxicity of inorganic particles and of assessment of ligand efficiency. Recent studies have shown that the cellular uptake of particles is difficult to detect experimentally. Quantitative measurements are still more challenging. Among the techniques used to evidence intracellular uptake, ITC should play a crucial role. Preliminary ITC experiments on hybrid core-shells have shown that thermodynamic experiments between living cells and nanostructures are realistic in terms of order of magnitude of binding enthalpy expected, sample volume and concentration needed.