Nanoparticles are one of the promising solutions for the current dilemma of drug transport to the central nervous system (CNS). The efficiency and specificity of treatments are compromised by the presence of cellular barriers that limit the passage of substances to the nervous tissue. Nanoparticles, and particularly dendrimers, are emerging as very promising tools for biomedical applications and envisioned as key players that will most likely have major roles in improving the means of diagnostics, imaging and therapeutics. However, despite the tremendous amount of literature involving NP synthesis, functionalization and in vitro and in vivo evaluation, basic knowledge on the interactions of these materials with the living systems is still sparse. The mechanisms of cellular internalization and clearance, intracellular trafficking and distribution, as well as degradation, remain unexplored. Yet, herein we investigated the interaction of dendrimer nanoparticles with astrocyte membranes using patch-clamp electrophysiology capacitance quantification and live confocal imaging. Carboxymethylchitosan/ poly(amido)amine (CMCht/PAMAM) dendrimer nanoparticles were developed recently in our lab and loaded with the corticosteroid methylprednisolone (MP). These dendrimer NP recently revealed a remarkable potential for administration and diffusion in the CNS, having inclusively showed interesting therapeutical properties in animal models of injury. In this study, we investigated how the NP interfered with the frequency of astrocyte membranar events, such as endocytosis and exocytosis vesicle formation. Primary astrocytic cultures were incubated with NP and: (i) prepared for electrophysiological readings; or (ii) labeled for endocytotic or exocytotic vesicles, for live confocal imaging. The patch clamp electrophysiology data revealed differences in the frequency of events arising in the astrocyte membrane in the presence of the nanoparticles, indicating that they interfere with the formation/ fusion of vesicles, both endocytic and exocytic. These alterations were more pronounced and significant regarding the formation of exocytotic vesicles. The live confocal imaging of both these types of vesicles confirmed that the nanoparticle trafficking in astrocytes involves these pathways, since co-localization of nanoparticles was seen both with endocytotic and exocytotic vesicles. We are directly showing for the first time that the MP-loaded CMCht/PAMAM dendrimer nanoparticles enter the endosomes and exosomes of astrocytes, meaning that after entering the cell the NP associate with exocytic vesicles to follow the exit route and be cleared from astrocytes. Nonetheless, the MP-loaded dendrimer NPs remain in the cells for a sufficient time period (about a week) to allow therapeutic intervention, while exiting the cells via exocytosis, in a dynamic way.