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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.

One of the most studied nanocarriers for drug delivery are polyamidoamine (PAMAM) dendrimers. However, the alterations produced by PAMAM dendrimers in neuronal function have not been thoroughly investigated, and important aspects such as effects on synaptic transmission remain unexplored. We focused on the neuronal activity disruption induced by dendrimers and the possibility to prevent these effects by surface chemical modifications. Therefore, we studied the effects of fourth generation PAMAM with unmodified positively charged surface (G4) in hippocampal neurons, and compared the results with dendrimers functionalized in 25% of their surface groups with folate (PFO25) and polyethylene glycol (PPEG25). G4 dendrimers significantly reduced cell viability at 1 µM, which was attenuated by both chemical modifications, PPEG25 being the less cytotoxic. Patch clamp recordings demonstrated that G4 induced a 7.5-fold increment in capacitive currents as a measure of membrane permeability. Moreover, treatment with this dendrimer increased intracellular Ca2+ by 8-fold with a complete disruption of transients pattern, having as consequence that G4 treatment increased the synaptic vesicle release and frequency of synaptic events by 2.4- and 3-fold, respectively. PFO25 and PPEG25 treatments did not alter membrane permeability, total Ca2+ intake, synaptic vesicle release or synaptic activity frequency. These results demonstrate that cationic G4 dendrimers have neurotoxic effects and induce alterations in normal synaptic activity, which are generated by the augmentation of membrane permeability and a subsequent intracellular Ca2+ increase. Interestingly, these toxic effects and synaptic alterations are prevented by the modification of 25% of PAMAM surface with either folate or polyethylene glycol.

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Vascular endothelial growth factor (VEGF) is highly expressed in vascular remodeling processes and accelerates reendothelialization after mechanical denudation. Two VEGF tyrosine kinase receptors have been reported—fms-like–tyrosine kinase-1 (Flt-1) and kinase domain region (KDR). Little is known about the regulation of the expression of these receptors after vascular injury. Herein, we have analyzed the expression of Flt-1 after mechanical denudation of primary cultures of endothelial cells, which has been considered a useful in vitro model to study endothelium responses to vascular injury. After denudation, the Flt-1 protein and mRNA levels are clearly up-regulated, and transient transfection experiments showed a strong induction of theflt-1 promoter-dependent transcription. Analysis of the flt-1 promoter sequence revealed the presence of a putative binding site for the early growth response factor-1 (Egr-1) at positions −24 to −16. Electrophoretic mobility shift and supershift assays showed that Egr-1 was able to bind to this DNA sequence, and cotransfection of the flt-1 promoter reporter plasmid with an Egr-1 expression vector resulted in enhancement of its transcriptional activity. Furthermore, the mutation of the Egr-1 binding site markedly reduced the denudation-induced flt-1promoter activity. These data demonstrate that Flt-1 is up-regulated after endothelial denudation and that Egr-1 plays a relevant role in this process.