Molecular modelling of TLR agonist Pam3CSK4 entrapment in PLA nanoparticles as a tool to explain loading efficiency and functionality

[Display omitted] Designing potent and safe-of-use therapies against cancers and infections remains challenging despite the emergence of novel molecule classes like checkpoint inhibitors or Toll-Like-Receptor ligands. The latest therapeutic perspectives under development for immune modulator administration exploits vectorization, and biodegradable delivery systems are one of the most promising vehicles. Nanoparticles based on Poly (D,L) Lactic Acid (PLA) as polymer for formulation are widely investigated due to its bioresorbable, biocompatible and low immunogen properties. We propose a PLA-based nanoparticle delivery system to vectorize Pam3CSK4, a lipopeptide TLR1/2 ligand and a potent activator of the proinflammatory transcription factor NF-κB that shows a self-assembling behavior from 30 µg/mL onwards. We demonstrate successful encapsulation of Pam3CSK4 in PLA nanoparticles by nanoprecipitation in a 40–180 µg/mL concentration range, with 99% of entrapment efficiency. By molecular modelling, we characterize drug/carrier interactions and conclude that Pam3CSK4 forms clusters onto the nanoparticle and is not encapsulated into the hydrophobic core. In silico predictions provide nanoprecipitation optimization and the mechanistic understanding of the particle dynamics. The loaded-Pam3CSK4 maintains bioactivity on TLR2, confirmed by in vitro experiments using reporter cell line HEK-Blue hTLR2. Our presented data and results are convincing evidence that Pam3CSK4-loaded in PLA nanoparticles represent a promising immune modulating system.