Mechanism of PAMAM Dendrimers Internalization in Hippocampal Neurons

Abstract

Polyamidoamine (PAMAM) dendrimers are hyperbranched macromolecules which have been described as one of the most promising drug nanocarrier systems. A key process to understand is their cellular internalization mechanism because of its direct influence on their intracellular distribution, association with organelles, entry kinetics, and cargo release. Despite that internalization mechanisms of dendrimers have been studied in different cell types, in the case of neurons they are not completely described. Considering the relevance of central nervous system (CNS) diseases and neuropharmacology, the aim of this report is to describe the molecular internalization mechanism of different PAMAM-based dendrimer systems in hippocampal neurons. Four dendrimers based on fourth generation PAMAM with different surface properties were studied: unmodified G4, with a positively charged surface; PP50, with a substitution of the 50% of amino surface groups with polyethylene glycol neutral groups; PAc, with a substitution of the 30% of amino surface groups with acrylate anionic groups; and PFO, decorated with folic acid groups in a 25% of total terminal groups. Confocal images show that both G4 and PFO are able to enter the neurons, but not PP50 and PAc. Colocalization study with specific endocytosis markers and specific endocytosis inhibitor assay demonstrate that clathrin-mediated endocytosis would be the main internalization mechanism for G4, whereas clathrin- and caveolae-mediated endocytosis would be implicated in PFO internalization. These results show the existence of different internalization mechanisms for PAMAM dendrimers in neurons and the possibility to control their internalization properties with specific chemical modifications.

Generation-6 hydroxyl PAMAM dendrimers improve CNS penetration from intravenous administration in a large animal brain injury model

“…On the other hand, internalization of nanoparticles by neurons only has been reported in few studies [43, 44]. Although in vitro study on hippocampal neuron showed clathrin mediated pathway as a main internalization pathway for G4 PAMAM dendrimers with amine terminal function groups [45], in vivo study of dendrimer uptake by neurons under pathological condition was rarely reported. In our model, hippocampal neurons receive a major glutamatergic input to hippocampus from the neurons in the entorhinal cortex (perforant pathway) [6, 35] and during ischemia-reperfusion are likely to exhibit abnormally high firing rates.…”

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation

“…An upper size limit reported for particles entering via this pathway is 200 nm [8, 11]. Silica NPs of 200 nm and polymeric NPs of 100–200 nm were reported to internalize predominantly through CME [11, 28], and the positive charge on the surface of quantum dots [29], dendrimers [30] and polymer NPs [10, 31] was shown to increase the probability of internalization via CME rather than the use of other endocytic pathway. In our study, the inhibitors of CME were not very successful to inhibit the uptake of Rubipy-SiO 2 NPs by CaCo-2 cells; however the effect of chlorpromazine and the colocalisation with clathrin suggests a minor role of this pathway in the internalization of 30 nm Rubipy-SiO 2 NPs.…”

Prevention of Synaptic Alterations and Neurotoxic Effects of PAMAM Dendrimers by Surface Functionalization

“…For applications in the central nervous system (CNS), different dendrimers have been used mainly to enhance the bioavailability, the specific targeting for this system and the ability to cross the blood-brain barrier of pharmacological agents [ 20 , 21 , 22 ]. It is also possible to mention specific studies related to internalization mechanisms of PAMAM dendrimers in neurons and glia cells [ 23 , 24 , 25 ]. In spite of the promising results obtained in in vitro and in vivo models for the treatment of glioma, cerebral palsy and neurodegenerative diseases [ 26 , 27 , 28 ], the biocompatibility and elimination of toxic effects of these polymers remain as a relevant challenge for their clinical application in CNS [ 3 , 29 ].…”