Mechanism of PAMAM Dendrimers Internalization in Hippocampal

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Mechanism Of PAMAM Dendrimer Internalization In Hippocampal Neurons Felipe Vidal, Pilar Vasquez, Carola Diaz, Daniela Nova, Joel Alderete, and Leonardo Guzman Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00381 • Publication Date (Web): 24 Aug 2016 Downloaded from http://pubs.acs.org on September 1, 2016

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Molecular Pharmaceutics

Mechanism Of PAMAM Dendrimer Internalization In Hippocampal Neurons Felipe Vidal a , Pilar Vásquez a , Carola Díaz b , Daniela Novaa , Joel Aldereteb , Leonardo Guzmán a*

a

Laboratory of Molecular Neurobiology, Department of Physiology, Faculty of Biological Sciences, University of Concepcion, Chile. b

Department of Organic Chemistry, Faculty of Chemical Sciences, University of Concepcion, Chile.

*

Corresponding autor: Leonardo Guzmán, Departamento de Fisiología, Facultad de

Ciencias Biológicas, Universidad de Concepción, Chile, Casilla 160-C. Tel.: 56412661229, Fax: 56-412245975

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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 its direct influence on their intracellular distribution, association to organelles, entry kinetics, and cargo release. Despite that internalization mechanisms of dendrimer 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 four generation PAMAM with different surface properties were studied: unmodified G4 with a positive charged surface, PP50 with a substitution of the 50% of amino surface groups with polyethylenglycol 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 inhibitors 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 mechanism for PAMAM dendrimers in neurons and the possibility of control their internalization properties with specific chemical modifications.



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Keywords: PAMAM internalization, dendrimer, endocytosis, neuronal uptake, surface modification.

Abbreviations: PAMAM, Polyamidoamine; G4, Fourth generation PAMAM dendrimer; PP50, Polyethylenglycol modified fourth generation PAMAM dendrimer, PAc, fourth generation PAMAM dendrimer modified with 2-carboxyethylacrylate; PFO, fourth generation PAMAM dendrimer modified with folic acid; FITC, Fluorescein isothiocyanate; CPZ, Chlorpromazine; Fil, Filipin complex.


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INTRODUCTION During the last several years, high throughput technologies and bioinformatics techniques have been developed for the generation of new drugs, but unfortunately old pharmacological problems such as solubility and bioavailability still remain. Moreover, in many cases, therapeutic targets are intracellular components, which means that pharmacological agents must be internalized by the cells. However, many new compounds do not always have the ability to cross the plasma membrane or other physiological barriers, making it necessary to improve their bioavailability. Nanocarriers systems have emerged as important tools to overcome the different limitations of small molecule, peptide and gene-based therapies

1-3

. Among the polymers used as nanocarriers, polyamidoamine

(PAMAM) dendrimers appear as one of the most promising

4, 5

. PAMAM dendrimers are

hyperbranched polymers organized from a central molecule of ethylenediamine that gives way to expansive growing layers, known as generations, terminating in a surface of primary amines that are positively charged at physiological pH 6. Their high stability, water solubility, well-defined size and composition, and an easily modifiable surface yield improved carrier properties making them the most successful nanocarriers used

4, 7, 8

. The

versatility of PAMAM dendrimers has demonstrated to be useful at improving the action of several important drugs related to anticancer, anti-inflammatory and antimicrobial treatment 9. Cellular internalization of PAMAM dendrimers have been studied and the results demonstrate that in general they are able to enter the cells by different endocytic mechanisms, among which clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and other mechanisms independent of clathrin and caveolae are the most prominent

10, 11 12, 13

.

. Different mechanisms of internalization mean different endocytic 5 ACS Paragon Plus Environment


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dynamics, intracellular trafficking and organelle association

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11, 14

. Clathrin-mediated

endocytosis is described as the major endocytic pathway and is commonly known as classic endocytosis where the vesicles fuse with early endosomes following an acidification route that finishes in lysosomes

15

. In the case of caveolae-mediated endocytosis, vesicles fuse

with Golgi or endoplasmic reticulum in a non-acidification alternative pathway, even when association with classic trafficking mechanisms has been observed 16. In addition, different molecules are internalized by receptor-mediated endocytosis through specific endocytic pathways

17

. For example, it is known that the transferrin receptor is internalized by

clathrin-mediated endocytosis

18

, whereas folate receptors are taken in by caveolae-

mediated endocytosis 19. These differences underscore the need to understand the details of dendrimer internalization mechanisms because of their influence on cargo distribution and intracellular release. The particular endocytosis mechanism of PAMAM dendrimers depends on their surface properties and the cell type. Thus, evidence shows that anionic dendrimers are internalized by caveolae-mediated processes, while the cationic and neutral dendrimers appear to be taken up by a caveolae- and clathrin-independent process in A549 lung epithelial cells

20

.

However, colocalization studies with specific endocytosis markers in HeLa cells show that the cationic dendrimers are internalized by clathrin-mediated endocytosis and micropinocytosis

21

. Moreover, the overexpression of caveolin-1, the main protein

component of caveolae, increases internalization of functionalized polyethylene glycol PAMAM dendrimers in HepG2 liver cells, while in C2C12 myoblast cells this overexpression does not influence the process

22

. On the other hand, in Caco-2 intestinal

cells, specific drug inhibition suggest that negatively charged surface PAMAM dendrimers enter the cell by clathrin- and caveolae-mediated endocytosis 23. 6 ACS Paragon Plus Environment

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In terms of the applications of dendrimers on the CNS, it has been important the development of dendrimers with the ability to target and pass through the blood-brain barrier (BBB) in neurons

28

13, 24-27

. However, although there are studies about dendrimer internalization

, further studies are needed to have a better understanding of the PAMAM

endocytic process

in

neuronal

cells.

Considering the relevance of improved

neuropharmacological therapies and the use of nanocarriers as an auspicious alternative to this aim, we examined the internalization of four PAMAM based dendrimers in hippocampal neurons from a molecular and mechanistic point of view, with the hypothesis that different modifications of their surface determine the internalization properties and pathways. Data from this study will provide a better understanding of nanocarrier entry kinetics, intracellular distribution, association to organelles and cargo release in a neuronal model.



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MATHERIAL AND METHODS Materials Fourth generation PAMAM dendrimers (G4, solved in methanol. Sigma CAS number: 163442-67-9), 550 Da polyethylene glycol, 2-carboxyethylacrylate, folic acid, fluorescein isothiocyanate (FITC), mouse anti-clathrin monoclonal antibodies, rabbit anti-caveolin-1, mouse anti-Rab5, mouse and rabbit anti-MAP-2, chlorpromazine hydrochloride (CPZ), filipin complex (Fil), FITC labeled transferrin and FITC labeled cholera toxin subunit B were purchased from Sigma-Aldrich.

Cell cultures Hippocampal neurons were obtained from 18-19 days C57BL/J6 mice embryos as previously described

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in accordance with NIH recommendations. The neuronal feeding

medium consisted of 90% minimal essential medium (MEM, BRL Technologies, Rockville, MD, USA), 5% heat-inactivated horse serum, 5% fetal bovine serum and a mixture of nutrient supplements. Cells were plated at 300,000 cells/mL and studied after 710 days in vitro (DIV).

Functionalization of PAMAM dendrimers Four different PAMAM dendrimers were used: (1) G4 PAMAM dendrimer (G4) that had 64 positively charged terminal groups; (2) G4 PAMAM that had 50% of its terminal groups modified with a polyethylene glycol molecule (PP50) following the Kojima et al. protocol 30

; (3) G4 PAMAM that was modified with 2-carboxyethylacrylate (PAc) by the Michael

Addition

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exposing negative charges on the surface in an average of 20 terminal groups

per molecule (covering approximately 30% of the terminal groups); and (4) G4 PAMAM 8 ACS Paragon Plus Environment

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that was modified with folic acid following the Benchaala et al. protocol

32

adding around

15 molecules to each dendrimer (covering approximately of 25% of the terminal groups). In addition, dendrimers were marked with FITC dye based on the Kolhe et al. protocol

33

linking an average of 4 molecules per dendrimer. Dialysis and washes were applied to the dendrimers. After a drying process, dendrimers were solved on PBS buffer (pH 7,4). Chemical modifications and absence of methanol and other reagents were verified by 1H NMR spectroscopy in a Bruker 400 Avance spectrometer (supplementary information).

Immunocytochemistry Hippocampal neurons were incubated with 0.5 µM FITC-labeled dendrimers for 30 minutes. After incubation, cells were washed with PBS solution and then fixed with 4% paraformaldehyde for 15 minutes. Cells were washed again and permeabilized with 0.1% Triton solution for 30 minutes. For endocytosis pathway studies, neurons were incubated for 16 hours with anti-clathrin (1:500), anti-caveolin-1 (1:1000) or anti-Rab5 (1:1000) antibodies as endocytosis mechanism markers, and anti-MAP-2 (1:200) antibody as a neuronal marker. Cy3 labeled anti-mouse or anti-rabbit antibodies (1:200) were used as secondary antibodies for endocytosis markers and AlexaFluor 647 labeled anti-mouse or anti-rabbit antibodies were used as secondary antibodies for MAP-2, incubating fixed cells during 1 hour. Finally, incubation for 10 minutes with a DAPI dye (1:300) for nuclei stain was performed.

Internalization analysis Hippocampal neurons were incubated for 30 minutes with 0.5 µM FITC-labeled G4 or PFO dendrimers. Subsequently, cells were washed with PBS solution to remove dendrimer 9 ACS Paragon Plus Environment


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excess and incubated with feeding media for 0, 2.5 or 5 hours. Cells were fixed, permeabilized and both neuronal and nuclei staining was performed as described in the immunocytochemistry protocol. For a quantitative analysis, a 2.2 x 3.96 µm region of interest (ROI) from the membrane to the inner neuronal region was established on different confocal images and a fluorescence plot profile was measured. To compare the different quantifications, the plot profile values for each ROI were normalized to the respective neuron fluorescence mean. Analysis was performed using MacBiophotonics ImageJ software.

Colocalization analysis Immunocytochemistry images were analyzed with MacBiophotonics ImageJ software. Manders’ coefficient

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per neuron was determined and averages were calculated to

determine the degree of colocalization of G4 and PFO dendrimers with different endocytosis markers.

Endocytosis inhibitors assay To test the endocytic pathways for each dendrimer, neurons were treated for 30 minutes with chlorpromazine (20 µg/mL), a clathrin-mediated endocytosis inhibitor, or filipin (2 µg/mL), a caveolae-mediated endocytosis inhibitor 20, 35. FITC-labeled transferrin was used as a clathrin-mediated endocytosis control, and FITC-labeled cholera toxin subunit B as a caveolae-mediated endocytosis control

35

(supplementary Fig. 2). Neurons were incubated

with 0.5 µM FITC-labeled G4 or PFO dendrimers for 60 minutes. Then, neurons were fixed and permeabilized as described in the immunocytochemistry protocol and incubated with an anti-MAP-2 antibody that specifically stains neurons and a DAPI dye that stains the 10 ACS Paragon Plus Environment

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nucleus. Fluorescence intensity of FITC-labeled dendrimers per neuron was measured with MacBiophotonics ImageJ software and compared with endocytosis inhibitor treated and non-treated neurons, considering the fluorescence mean of non-treated cells as 100% of the fluorescence intensity.

Confocal microscopy Imaging for the different assays was obtained on a spectral confocal microscopy (LSM780 NLO, Zeiss) equipped with a Plan-Apochromat 63x / 1.40 Oil DIC M27 objective and 405 nm, 488 nm, 561 nm and 633 nm lasers. Fluorescence detection equipment consisted of a polychromatic detector with 32 GaASP and 2 PMP FL photomultipliers and a T-PMT detector for transmitted light. Images were obtained at the Centro de Microscopía Avanzada (CMA Bio-Bio) at the University of Concepcion, Chile.

Statistical analyses All experiments were performed using for convenience non-probability sampling, with each neuron of three different extractions as the study unit. In the case of endocytosis inhibitors assay, Levene and Shapiro-Wilk tests were used for the evaluation of homogeneity of the variances and normality of the variables, respectively. Kruskal-Wallis test was performed to evaluate significant differences between inhibitors treated and control neurons.



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RESULTS Internalization of different PAMAM dendrimers The surface characteristics of PAMAM dendrimers are a key property for their cellular internalization ability 36. In this study, we examined the cellular internalization capacity of four different PAMAM based dendrimers: G4 PAMAM (G4) had a positively charged surface; G4 PAMAM modified with polyethylene glycol (PP50) to incorporate neutral groups; G4 modified with 2-carboxyethylacrylate anionic groups (PAc); and G4 PAMAM modified with folate groups (PFO) (figure 1). Hippocampal neurons were incubated for 30 minutes with different FITC-labeled dendrimers and analyzed using confocal microscopy. The image analyses showed that G4 and PFO dendrimers were able to enter the cells, whereas PP50 and PAc remained outside the neurons (figure 2A). Because the internalization of G4 and PFO at 30 minutes of incubation shows a peripheral distribution of dendrimers and it could be considered as a short time to discard the uptake of PP50 and PAc, longer times of incubation were performed. At 6 hours of incubation, is possible to observe an important amount of G4 and PFO fluorescent mark in the cytoplasm of neurons which is distributed not only in the periphery, but also in more internal regions. For the case of PP50 and PAc even at these time it was not possible to observe their fluorescent mark on neurons (figure 2B). To explore if these dendrimers have slower uptake dynamics, neurons were incubated for 12 hours with these dendrimers, but no internalization was observed (supplementary information).

G4 and PFO dendrimer internalization at different times After observing an early internalization of G4 and PFO dendrimers, we decided to study the sequence of this internalization process in a more detailed manner. Incubation with 12 ACS Paragon Plus Environment

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FITC-labeled G4 and PFO dendrimers for 30 minutes in neurons showed a mainly peripheral distribution on the cells, particularly at the soma (figure 2A). In order to follow the internalization dynamics, after 30 minutes incubation with G4 or PFO, neurons were washed to remove the dendrimer excess and maintained in culture medium. Images were taken immediately after the wash (0 h), 2.5 and 5 hours later. The results showed that at time 0 the fluorescent label of dendrimer was located in the periphery of the neurons, as previously demonstrated. Interestingly, at 2.5 and 5 hours it was possible to see a marked change in fluorescence distribution to a more internal cell region (figure 3A and 3C). A more detailed quantification of the fluorescent distribution demonstrated that dendrimers have a differentiated distribution pattern depending on the time elapsed from initial incubation. The fluorescence peak displaced to more internal locations in the soma with the course of the internalization process (figure 3B and 3D). These observations suggest that G4 and PFO dendrimers are able to rapidly interact with the neuronal surface and be internalized and concentrated in a granular manner.

Colocalization with specific endocytosis markers To determine the internalization mechanism involved in hippocampal neurons, colocalization analyses of G4 and PFO dendrimers with specific endocytosis markers were performed. Hippocampal neurons were exposed to FITC-labeled G4 and PFO dendrimers and analyzed with immunocytochemistry using anti-clathrin, anti-caveolin-1 or anti-Rab5 antibodies as markers for clathrin-mediated endocytosis 16

15

, caveolae-mediated endocytosis

or early endosome and classic route trafficking markers

37

, respectively. Confocal

analyses showed a relevant colocalization of G4 with clathrin and Rab5 but not with caveolin-1 (figure 4A). Images were analyzed and Manders’ coefficient was determined for 13 ACS Paragon Plus Environment


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the different markers to quantitate the colocalization with G4 dendrimers (figure 4B)

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34

,

obtaining values of 0.443 ± 0.053 for clathrin, 0.254 ± 0.119 for caveolin-1 and 0.473 ± 0.117 for Rab5. These results were in agreement with the qualitative observations, showing a poor colocalization with caveolin-1 marker and higher values for clathrin and Rab5 markers indicating that clathrin-mediated endocytosis and classic intracellular trafficking routes are important internalization mechanisms for G4 dendrimers in hippocampal neurons. Following the same experimental scheme, data demonstrated that PFO colocalized with the caveolae marker, and at the same time, it was possible to observe colocalization with clathrin and Rab5 markers (figure 4C). The data was supported by Manders’ coefficients that revealed an important degree of colocalization with the three markers, however, it is important to mention that the higher value was obtained for caveolin-1 with 0.555 ± 0.062, whereas the values for clathrin and Rab5 were 0.407 ± 0.106 and 0.470 ± 0.055, respectively (figure 4D). These results suggest that folic acid groups induce the internalization of PFO dendrimers mainly by a caveolae-mediated endocytic process, but also with participation of clathrin-mediated endocytosis.

Endocytosis inhibitors assay. To complement these results, the effect of specific endocytosis inhibitors on G4 and PFO internalization was studied. Chlorpromazine and filipin were used as clathrin- and caveolae-mediated endocytosis inhibitors, respectively

35

. Confocal images were obtained

and the FITC-labeled dendrimer fluorescence in neurons was measured. The results for G4 dendrimers showed a significant reduction to a 53.7% (Q1: 38.3%; Q3: 63.1%) of internalization in neurons when cells were treated with chlorpromazine, indicating that 14 ACS Paragon Plus Environment

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these dendrimers follow the clathrin-mediated endocytosis in this cell type (figure 5A). On the other hand, treatment with filipin did not show a reduction in G4 internalization, obtaining a 93.4% (Q1: 81.1%; Q3: 109.6%) value indicating that caveolae-mediated endocytosis is not an important process for the internalization of these dendrimers in neurons (figure 5B). For the PFO assays, the treatment of neurons with both inhibitors demonstrated a significant reduction in its internalization (figure 5C), specifically diminishing to 55.8% (Q1: 51.13%; Q3: 62.9%) for chlorpromazine treatment and 47.6% (Q1: 38.6; Q3: 68.0%) for filipin treatment, suggesting that clathrin- and caveolae-mediated endocytosis are both implicated in the internalization process of these dendrimers in neurons (figure 5D). These results agree with colocalization assays and suggest that neuronal internalization mechanisms of dendrimers are mainly by clathrin-mediated endocytosis in the case of G4, whereas PFO enters the neurons by both clathrin- and caveolae-mediated endocytosis. The latter result suggests that functionalization of G4 dendrimers with folic acid molecules is able to direct in part their internalization through caveolae-mediated endocytosis.



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DISCUSSION Since PAMAM dendrimers were developed as polymeric drug nanocarriers 38, they have been used as an important tool for the improvement of pharmacological treatment and gene therapies with promising results

4, 39

. The great number of surface positive charges have

been associated to cytotoxic effects, but this aspect has been resolved by chemical modifications with groups as polyethyleneglycol and pyrrolidone, among others

40, 41

.

Several studies have demonstrated the ability of these compounds to reach intracellular regions in different cell types by endocytic processes

20-23

. In this study, we examined the

particular internalization mechanism of PAMAM dendrimers in neurons and analyzed the possibility of specifically directing the process using surface modifications. According with our results, hippocampal neurons internalized both G4 and PFO, but not PP50 and PAc dendrimers, which is in agreement with previous reports. For example, a study in A549 lung epithelial cells showed that PAMAM dendrimers with a complete neutral or negative surface had a lower degree of internalization compared with those having a positive surface in flow cytometry assays

20

. Moreover, a study of GATG

dendrimer internalization revealed a strong internalization of cationic dendrimers, but a weak internalization for anionic and no internalization for neutral dendrimers in confocal microscopy assays

42

. Our results showed that a partial modification of positive PAMAM

surface had a strong influence on their internalization properties in hippocampal neurons. The substitution of the 50% surface amino groups with polyethylenglycol molecules reduces to the half the surface charge density of dendrimers. In the case of acrylate modifications, exist a reduction of surface charge density for the replacement of amino groups in a 30%, but also a reduction of other 30% due to the neutralization of positive charges by negative ones present in acrylate groups. This implicates that PP50 and PAc 16 ACS Paragon Plus Environment

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dendrimers have a similar total surface charge density and explain their comparable inability to neuronal uptake. The fact of a partial modification of dendrimer surface outcomes in a complete blockade of their internalization could be determined for the intrinsic endocytic properties of neurons. Future studies could be compare the endocytic dynamics of these dendrimers in neurons with the process in other cell types to explore this aspect. In terms of internalization mechanisms, here we demonstrate that G4 dendrimers enter the neurons mainly through a mechanism that involves clathrin-mediated endocytosis, which proceeds to the classic intracellular trafficking as indicated by colocalization with clathrin and Rab5 markers. However, the results suggest that this would not be the exclusive internalization mechanism because the FITC fluorescent label was not completely associated to those markers. Moreover, the chlorpromazine treatment did not completely inhibit the G4 internalization. On the other hand, these results discard caveolae-mediated endocytosis as a possible G4 internalization pathway in neurons, even when other internalization mechanisms independent of clathrin and caveolae could be involved. Interestingly, PFO-conjugated PAMAM was capable of being internalized through both clathrin- and caveolae-mediated endocytosis. This suggest that folic acid groups would be leading the internalization of these dendrimers by interaction with folic acid repector, which follows a caveolae-mediated endocytosis. However, is not possible to observe a complete change of endocytosis pathway comparing with unmodified G4. An explanation of this fact is the low percent of substitution with folic acid groups (15 molecules out of a total of 64 amine groups), where the important number of positive charges that still remains on the dendrimer surface leads the internalization through clathrin-mediated endocytosis like the case of G4. Studies with higher degrees of functionalization are needed to 17 ACS Paragon Plus Environment


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corroborate this possibility, although these results demonstrate that surface modification with specific groups allows to direct the internalization route of dendrimers for a desired purpose. Knowledge of the endocytosis mechanisms of dendrimers is relevant for the possibility to specifically direct them towards particular applications. It is widely known that the conformation of these polymers is modulated by pH which has a key influence on drug delivery 1, 5. Considering this, reduced pH in the classic endocytosis pathway may favor the release of small drugs encapsulated in the inner cavities of dendrimers, or cleave the covalent bond of molecules linked to its surface. In relation with gene transfection, an important concern is to avoid the degradation of nucleic acids by lysosomes associated with classic endocytosis

43

. Superficial modifications of dendrimers with folate groups allowed

for internalization that was able to evade the intracellular degradation machinery achieving an enhanced transfection efficiency in HeLa and HepG2 cells 44, 45. CNS diseases have always been an important field of study because of their detrimental effect on the organism and also because of the increasing incidence in this group of pathologies

46

. Although CNS-directed use for dendrimer application has not been well

studied, in the last years important results have been found. One remarkable example is that encapsulated siRNA in a PAMAM-based nanocarrier which showed efficient knockdown of beclin-1 demonstrating the autophagy role in NMDA effects in rat cortical neurons

47

.

Additionally, it was determined that PAMAM dendrimers used for the delivery of N-acetylL-cysteine (NAC) suppresses neuroinflammation and leads to a marked improvement in motor function in a rabbit cerebral palsy model 48. Therefore, an important field of study is dendrimer modifications for CNS targeting and the pass through BBB, which has already demonstrated effectiveness as a nanocarrier systems

26, 27


. For example, T7 peptide

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functionalized dendrimers loaded with the antitumor gene agent pORF-hTRAIL and doxorubicin, target the transferrin receptor and induce a three times survival increase in a mice brain glioma model

49

. Moreover, functionalized dendrimers with peptides derived

from Kunitz domains (Angiopep-2, specific for LRP-1 receptor) are capable of efficiently transporting gene encoding neurotrophic factor (hGDNF) derived from a human glial cell line having neuroprotective effects in a Parkinson’s rat model 50. In summary, our results contribute to the understanding of different potential CNS uses for PAMAM dendrimers having a known intracellular route and their influence on cargo delivery allowing for the development of future applications. In particular, high interest goals are the modulation of synaptic activity, higher neural functions, behavioral studies and related pathologies such as epilepsy, schizophrenia, neurodegenerative diseases and drug abuse addiction. Neuronal internalization ability, description of intracellular trafficking and the possibility of specific subcellular targeting of dendrimers are relevant aspects to advance towards new neurological applications.



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CONCLUSIONS One of the fundamental processes to understand the biological action of dendrimers as drug carriers, is its mechanism of cellular uptake. Although advances have been reported in this area, very little is known about the characteristics that has the endocytosis of PAMAM dendrimers in neurons. One of the most interesting features of PAMAM dendrimers is the ability to be covalently modified at their terminal groups. Thus, in this study we analyzed the capacity of internalization of hippocampal neurons of four types of dendrimers based on fourth generation PAMAM; unmodified (G4), modified with polyethylenglycol (PP50), with acrylate (PAc) or folate (PFO). Through analysis of confocal images it was determined that only the dendrimers G4 and PFO are internalized by neurons, whereas PP50 and PAc dendrimers have no capacity to be internalized. Through immunocytochemistry and inhibitors studies it was determined that endocytosis of G4 and PFO are associated with clathrin-mediated endocytosis. PFO is also associated with both caveolae-mediated endocytosis. In addition, both internalized dendrimers associate with Rab5, an early endosome marker. According to the above, the hypothesis indicating that different chemical surface modifications of dendrimers generate different internalization properties is demonstrated.


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ACKNOWLEDGEMENTS Authors thank to Centro de Microscopía Avanzada CMA Bio-Bio for microscopy imaging, Mrs. Lauren Aguayo and Cesar Lara for technical assistance. This work was supported by CONOCYT - FONDECYT grant 1131004 (Leonardo Guzman) and 1130531 (Joel Alderete). Felipe Vidal and Carola Díaz thanks CONICYT for doctoral fellowships.



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Figure 1. Schematic representation of dendrimers. Four different PAMAM dendrimers were studied. G4, fourth generation PAMAM dendrimer with a positive charged surface (red). PP50, G4 surface modified with 50% polyethylene glycol neutral groups (grey). PAc, G4 modified with 2-carboxyethylacrylate (blue) which exposes negative charges on the surface. PFO, G4 modified with folic acid (purple). All of the dendrimers were conjugated with FITC dye (green) for the study. 182x96mm (150 x 150 DPI)

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Figure 2. Internalization study of different dendrimers in neurons. Hippocampal neurons were incubated with different FITC-conjugated dendrimers (green). MAP2 (white) was used to mark neurons and DAPI (blue) for nucleus. A) Neurons were incubated 30 minutes with the indicated dendrimer (0.5 µM), washed and processed for immunocytochemistry. Both normal confocal (top panel) and transmitted light (bottom panel) images are shown for a better visualization of the internalized molecules. B) Neurons were incubated for a period of 6 hours with the indicated dendrimers (0.5 µM) following the same protocol as in A). Scale bar = 10 µm. 164x198mm (150 x 150 DPI)


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Figure 3. Internalization at different times. After incubation with dendrimers, the solution was removed and neurons were incubated with culture medium for different times before imaging. A) Images of the G4 fluorescent dendrimer were obtained at 0, 2.5 and 5.0 h after incubation as indicated. B) Fluorescence intensity (relative fluorescence units, RFU) distribution at different times after G4 incubation considering the rectangle area marked in a membrane to nuclei direction as described in Methods section. C) Images of the PFO fluorescent dendrimer were obtained at 0, 2.5 and 5.0 h after incubation as indicated. D) Fluorescence intensity (relative fluorescence units) distribution at different times after PFO incubation considering the rectangle area marked in a membrane to nuclei direction as described in Methods section. Scale bar = 5 µm. 168x132mm (150 x 150 DPI)



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Figure 5. Determination of endocytosis mechanisms using specific inhibitors. Previous to incubation with FITC-labeled G4 and PFO dendrimers, hippocampal neurons were treated with chlorpromazine (CPZ) as a specific inhibitor for clathrin-mediated endocytosis and filipin (Fil) as a specific inhibitor for caveolaemediated endocytosis. Dendrimer incubation without previous inhibitor treatment was used as control. The fluorescent mark associated to dendrimers was measured in each neuron area to evaluate the level of internalization. A) Images of G4 internalization profile without inhibitor treatment (left column) and under treatment with CPZ (middle column) and Fil (right column). B) Quantification of the intensity of fluorescence as percentage from control expressed in median as measure of central tendency and quartile range as a measure of dispersion. C) Images of PFO internalization profile without inhibitor treatment (left column) and under treatment with CPZ (middle column) and Fil (right column). D) Quantification of the intensity of fluorescence as percentage from control expressed in median as measure of central tendency and quartile range as a measure of dispersion. Scale bar = 5 µm. 203x181mm (150 x 150 DPI)


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Mechanism of PAMAM Dendrimers Internalization in Hippocampal

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