Thesis presented June 28, 2021

Abstract:
Grafting of prodrugs on cellulose nanofibrils (CNF) has emerged as an interesting route for the design of controlled-release-carriers in drug delivery applications. However, in-depth chemical and structural characterization of the CNF surface chemistry is often an unmet challenge that goes beyond the current sensitivity and resolution limits of standard characterization techniques, especially for low weight percentage (< 5 wt%) of functionalization as often obtained when using green chemistry routes.
In this work, we used Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP) to overcome the sensitivity limitation of solid-state NMR and to investigate surface chemistry of CNF functionalized with different prodrugs (metronidazole and ciprofloxacin). We notably showed that MAS-DNP is the only technique that can unambiguously probe the surface of these systems in case of very low functionalization (< 1 wt%) and provide answers to the three following major questions: degree of functionalization, differentiation between bound and adsorbed chemical species, and purity of the final product, in only ~ 2 hours of experimental time. DNP helped to point out the presence of residual coupling agents in the final product, which were previously believed to be removed by conventional washing procedure. We present a comparative study of CNF functionalized with different model compounds and prodrugs to evaluate the efficiency of two coupling agents (EDC/NHS vs. DMTMM). We show how the prodrug itself can affect the mode of fixation and the amount of loaded drug, highlighting the importance of pH during washing steps to avoid unwanted hydrolyzation of the prodrug linker and ensure efficient removal of unwanted chemical species (coupling agents, adsorb prodrug or side products of reaction).
Additionally, the sensitivity gain provided by DNP combined with the use of CNF-functionalized ¹⁵N-enriched spy molecules (benzylamine) allowed us to probe the surface of TEMPO-oxidized CNF with unprecedented details. We could thus characterize accessible and non-accessible cellulose chains and provide insight into the orientational geometry of both grafted and adsorbed benzylamine molecules on the CNF surface.
This work finally presents a study on the secondary cell-wall of hardwood sample from a matured Japanese beech plant in its native state at natural isotopic abundance. We present a novel DNP sample preparation, which made it possible to record ¹³C-¹³C 2D correlation experiments in only ~ 51 hours of experimental time. We provide, for the first-time, a direct insight into the complex atomic structure of plant secondary cell-wall currently beyond the reach of any other existing structural investigation tool. The atomic scale structural understanding of native biomass is essential, e.g., for their efficient conversion into biofuels.
We believe this thesis can serve as a base for the development of MAS-DNP in the routine characterization of surface functionalized nanocellulose materials and drug carriers, including the in-depth structural investigation of their surface. It demonstrates as well the potential of MAS-DNP and the important role it could play in unraveling the structural mysteries of na-tive plant cell-wall at the atomic scale.

Keywords:
DNP, Magic Angle Spinning, Secondary cell-wall, nanocellulose, hardwood, NMR