Therapeutic lipid nanoparticles in a biomimetic medium: transformation and interactions

The fate of nanomedicines in biological media remains poorly understood, particularly in terms of stability, structural evolution and interactions with various biological components. As a result, this lack of comprehension impedes their clinical use.
This thesis proposes to study the behavior of therapeutic nanoparticles (NPs) formed through the self-assembly of Leu-enkephalin-squalene (LENK-SQ) bioconjugates in media mimicking blood. Developed as an alternative to conventional opioids for the treatment of moderate to severe pain, these NPs exhibit greater analgesic activity than free Leu-enkephalin (LENK) after intravenous administration in murine models.
The morphological and structural changes undergone by these NPs in biomimetic media have been characterized, along with their interactions with several model blood proteins, including bovine serum albumin (BSA). These characterizations relied on a combination of complementary techniques, namely small-angle X-ray and neutron scattering (SAXS and SANS), and cryogenic transmission electron microscopy (cryo-TEM).
First, the formulation of the LENK-SQ NP suspensions by nanoprecipitation was investigated. The influence of various parameters on the properties of the resulting suspensions was assessed, and several potential issues were identified, highlighting the importance of thorough physicochemical characterization. Second, the self-assembly of the bioconjugates, initially organized into sponge phase NPs, was shown to evolve in response to changes in pH and ionic strength, with a transition towards wormlike micelles. These findings raise questions about the supramolecular organization adopted by LENK-SQ assemblies following injection and open up new perspectives for the design of more sophisticated formulations that could optimize drug delivery.
Finally, incubation of LENK-SQ self-assembled systems (micelles or NPs) with proteins revealed the formation of micelle-protein aggregates. Their characterization is consistent with structures formed through the association of proteins at the surface of LENK-SQ micelles.
Overall, this study reveals the complexity of the phenomena driving the interactions between soft nano-assemblies and blood proteins. This complexity arises from the coexistence of multiple species in dynamic equilibrium within the mixture. In the present case, these species include NPs, micelles, monomeric bioconjugates, proteins as well as micelle-protein aggregates.
In conclusion, this work highlights the importance of thorough physicochemical characterization of soft therapeutic nano-assemblies, which is likely to provide crucial information leading to a better understanding of their action mechanism.