Unveiling the intracellular trafficking of drug-loaded dendrimer nanoparticles in astrocytes

Introduction: Multifunctional dendrimers are emerging as promising solutions to the current dilemma of drug delivery to the central nervous system (CNS). Herein, we are proposing the use of carboxymethylchito- san (CMCht)-grafted PAMAM dendrimer nanoparticles (NP) to provide a targeted and sustained methylprednisolone (MP) release to CNS cells. MP is widely used following neurotrauma and neuroinflammation, however its actions are accompanied by deleterious effects. Despite recent research on nanomedicine, basic knowledge on the interactions of NP with living systems is still sparse. Thus, the interaction of MP-NP with the astrocyte membrane and its intracellular trafficking were investigated using a new combination of techniques, namely patch-- clamp electrophysiology and live confocal imaging.

Materials and methods: MP-loaded CMCht/PAMAM NP were synthe- sized as reported.1,2 Primary astrocyte cultures were prepared from P3 Wistar rat newborns. For electrophysiology readings, cell-attached high resolution membrane capacitance recordings were performed with a two-phase lock amplifier (SWAMIIB, Celica).3 Co-localization studies in live astrocytes were done incubating AlexaFluor"546 Dextran for endo- some labeling, and transfecting mCherry-Neuropeptide Y for exocytotic vesicle observation. Statistical significance was assessed by one-way ANOVA with Bonferroni post-test.

Results: Differences in the frequency of astrocyte endocytosis and exo- cytosis were observed in the presence of NP (Fig. 1). These differences were more pronounced in the formation of exocytotic vesicles. More- over, acute exposure to NP revealed larger endosomes, while the exocy- tic vesicles had smaller diameters following NP incubation. Live confocal imaging confirmed NP trafficking in astrocytes involves these pathways, since co-localization of NP was observed associated to endo- cytotic and exocytotic vesicles. Prolonged exposure of NP for a week revealed continued co-localization with exosomes, while almost negligi- ble endosomal transport.

Discussionandconclusions: Itwasconfirmedforthefirsttimetheend- ocytic and exocytic transport of MP-loaded CMCht/PAMAM dendrimer NP. More importantly, NP were shown to be recycled and continuosly cleared out from astrocytes via exocytosis one week after incubation, unveiling its intracellular trafficking pathways.

Acknowledgments: Portuguese Foundation for Science and Technology (S.R.C.SFRH/48406/2008; J.M.O.IF/00423/2012); European Science Foundation COST STSM.
Disclosures: The authors have nothing to disclose.

References
1. Oliveira JM, et al. Advanced Functional Materials 18(12):1840, 2008.
2. Cerqueira SR, et al. Small 9(5):738, 2012.
3. Rituper B, et al. Nature Protocols 8(6):1169,2013.