Exploiting Bacterial Pathways for BBB Crossing with PLGA Nanoparticles Modified with a Mutated Form of Diphtheria Toxin (CRM197): In Vivo Experiments

Abstract

Drugs can be targeted to the brain using polymeric nanoparticles (NPs) engineered on their surface with ligands able to allow crossing of the blood-brain barrier (BBB). This article aims to investigate the BBB crossing efficiency of polymeric poly lactide-co-glycolide (PLGA) NPs modified with a mutated form of diphtheria toxin (CRM197) in comparison with the results previously obtained using PLGA NPs modified with a glycopeptide (g7-NPs). Different kinds of NPs, covalently coupled PLGA with different fluorescent probes (DY405, rhodamine-B base and DY675) and different ligands (g7 and CRM197) were tested in vivo to assess their behavior and trafficking. The results highlighted the possibility to distinguish the different kinds of simultaneously administered NPs and to emphasize that CRM-197 modified NPs and g7-NPs can cross the BBB at a similar extent. The analysis of BBB crossing and of the neuronal tropism of CRM197 modified NPs, along with their BBB crossing pathways were also developed. In vivo pharmacological studies performed on CRM197 engineered NPs, loaded with loperamide, underlined their ability as drug carriers to the CNS.

PLGA-PEG-ANG-2 Nanoparticles for Blood–Brain Barrier Crossing: Proof-of-Concept Study

“…In analyzing the images, Ang-2-NPs colocalized with the various cell types present in the brain, evidenced only with DAPI, but were often also in close proximity to the neuronal cells ( Figure 5B, red and yellow arrows respectively). This is interesting because it could indicate a different mode of cell uptake than what has been seen previously for PLGA NPs targeted with the simil-opioid peptide ligand g7 [20,27,33] that are broadly up-taken only by neurons. Moreover, further studies will be required to better elucidate the mechanism of Ang-2 NP entrance in the brain or in the cells, for instance by blocking endo-transcytosis or the clathrin/caveolin uptake Pharmaceutics 2020, 12, 72 8 of 11 process, and therefore to draft a complete hypothesis on BBB-crossing pathways and neuron uptake of these kinds of NPs.…”

“…Moreover, literature extensively reported its ability to encapsulate, protect, and deliver a wide range of bioactive compounds (small molecules, proteins, enzymes, etc.) [20][21][22]. However, only a few studies indicated that PLGA-based nanoparticles modified with Ang-2 could be a promising active brain-targeting drug delivery system [13,14,18,23,24].…”

Intracellular trafficking of diphtheria toxin and its mutated form (CRM197) in the endocytic pathway

“…Drugs delivered by CRM197 carrier protein from endothelial cells which form blood-brain barrier are directed from apical towards basal lobes, thus delivery of drugs into brain tissue is achieved through transcytosis has been accepted. Delivery of lopermide into neurons was observed with in vivo studies by means of the strategy of overpassing blood-brain barrier using CRM197 nanoparticles [ 23 ].…”

Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro

“…Despite the controversy and murine DT-resistance, mouse-models are frequently used to investigate the function of CRM197 as vaccine carrier (Stickings et al 2008;Fiorino et al 2012;Mishra et al 2018) or as a coating on nanoparticles to mediate blood-brain barrier crossing (Tosi et al 2015). Therefore, we re-investigated binding to cell surface and internalization into DT-resistant murine cells using novel technologies, namely super-resolution optical microscopy giving us qualitative and quantitative insight into receptor binding and cellular uptake of different nontoxic DT mutants (e.g.…”