Gene delivery by dendrimers operates via a cholesterol dependent pathway

Abstract

Understanding the cellular uptake and intracellular trafficking of dendrimer-DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer-DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.

Figure 1

Cholesterol depletion reduces transfection efficiency. Cells were transfected with plasmids encoding either (A) EGFP or (B and C) β-galactosidase. Transfections were carried out following cholesterol chelation with MβCD, nystatin or filipin III. Transfection efficiencies were evaluated by (A) flow cytometry, (B) X-Gal staining or (C) ONPG colometric assay. All the experiments were carried out in triplicate. Error bars represent the standard error of the mean: (A) n = 4; (B) n = 2. Statistical significance (unpaired t test) is represented as follows: ***, p < 0.05; **, p < 0.1; *, p < 0.2 (A and B). The error bars of a representative experiment in (C), expressing the amount of β-galactosidase protein produced, correspond to the standard deviation of the mean.

Figure 2

Transfection efficiencies of cholesterol depletion on CHO and HEK 293 cells. A representative experiment shows the effect of cholesterol depletion with MβCD on the transfection efficiency on (A) CHO and (B) HEK 293, as previously shown in Figure 1 for EA.hy 926 cells. The cells were transfected using EGFP reporter gene and triplicate wells were pooled together prior to flow cytometry.

Figure 3

The effect of reconstitution of cholesterol on gene delivery. (A) The transfection efficiency using pEGFP (n = 6); (B) transfections carried out using pCMVβ-gal (n = 4). Cells were treated with MβCD alone (10 mM), MβCD with soluble cholesterol at either 0.25 or 0.5 mg/ml or left untreated (untr). Following treatment, the cells were transfected with dendriplexes and the transfection efficiency determined as described in Figure 1. Error bars represent the standard error of the mean. Statistically significant differences (unpaired t test) are shown as follows: ***, p < 0.05; *, p < 0.2. Following MβCD-depletion, the cholesterol pool was restored (C) by adding 0.4 mg/ml of either cholesterol (water-soluble, SC) or 5-cholesten-3β-ol (water insoluble, ISC). This latter source was not available for restoration. The results are presented as the mean percentage of replenishment compared with untreated cells (control) ± the standard error of the mean of three independent experiments.

Figure 4

Post-transfection MβCD-depletion and gene expression. The figure shows the effect of MβCD treatment of cells 1 h prior to transfection and 24 h after transfection with dendriplexes formed using either (A and B) pEGFP or (C and D) pCMV-βgal. At 48 h the transfection efficiency was evaluated as previously reported. The result shows a representative experiment where error bars represent the standard deviation of the mean.

Figure 5

Kinetics of cellular uptake of dendrimers in the presence or absence of MβCD and cell viability at different temperatures. Cells were untreated (left panel) or treated with MβCD (right panel) and then incubated at 37°C (closed symbols) or 4°C (open symbols) with FITC-labelled dendriplexes. The cells, after washing off the unbound complexes, were analysed by flow cytometry at various times to determine the amount of dendriplexes both bound to the cell surface and internalized. The results are plotted as mean ± SEM of four and three independent experiments in untreated cells and MβCD-treated cells, respectively.

Figure 6

Internalization of fluorescein-labelled dendriplexes. (A) Cells were incubated with complexes formed using FITC-conjugated dendrimers at 4°C for 15 min to prevent internalization. The samples were then warmed to 37°C and, at various times, the amount of internalized dendriplexes was determined after removing cell-surface-bound material with 5 mM NaOH. The results represent the mean ± SEM of three independent experiments of both untreated and cholesterol-depleted cells. (B) Dendriplexes formed with Cy3-dendrimers (green) and Cy5-DNA (red) were incubated with untreated or MβCD-treated EA.hy 926 cells for 15 min or 3 h as indicated. The cells were fixed and stained with DAPI (nuclear stain, blue) and then examined by confocal microscopy.

Figure 7

Co-localization of dendriplexes and ganglioside GM1. (A) EA.hy 926 cells were either untreated or treated with MβCD followed by transfection with dendriplexes formed using FITC-dendrimers. Cells not incubated with dendriplexes were used as a control. Cells were then probed with anti-FITC beads and lysed. This lysate was run through a column retaining the beads. Total lysate (T), the unbound material (UB) and the bound fraction (B) were then spotted onto the nitrocellulose. Dot blots were probed with either HRP-conjugated ChTxB, for detecting GM1 ganglioside, or anti-FITC antibody to localize dendriplexes. Data show that GM1 as well as dendrimers were both detected in the total lysates and the fractions bound to the columns of both untreated and MβCD treated cells. A weak signal for both GM1 and FITC-dendrimers was also detected in the unbound fraction of untreated cell lysate. In control untransfected cell lysates, only dots of total and unbound fraction, but not the bound fraction, were positive for GM1, whereas no FITC-dendriplexes were detected. (B) Confocal microscopy was performed on living cells that had been incubated with dendriplexes formed using Cy3-dendrimers (red) and Cy5 DNA (blue), together with FITC-ChTxB (green) which binds to the raft marker ganglioside GM1. (C) In addition, cells were imaged that had been incubated with Cy3 dendrimers alone (no DNA) together with FITC-ChTxB.