Lipoplex-mediated Transfection of Mammalian Cells Occurs through the Cholesterol-dependent Clathrin-mediated Pathway of Endocytosis

Abstract

Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-␤-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas cell-association remained unaffected. These data strongly support the notion that complex internalization, rather than the direct translocation of plasmid across the plasma membrane, is a prerequisite for accomplishing effective lipoplex-mediated transfection. We demonstrate that internalized lipoplexes colocalize with transferrin in early endocytic compartments and that lipoplex internalization is inhibited in potassium-depleted cells and in cells overexpressing dominant negative Eps15 mutants. In conjunction with the notion that caveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesteroldependent clathrin-mediated pathway of endocytosis.Currently, several carrier systems, including those based on synthetic cationic amphiphiles, are exploited for delivery of DNA constructs into cells for cell biological or therapeutic purposes. Compared with viral vectors, the transfection efficiency with most of the amphiphilic carriers ("lipoplexes") is still relatively low. However, because the latter offer considerable advantages over the former in terms of biological inertness, health risks, and large scale production, efforts are ongoing to improve their effectiveness of delivery. To achieve this goal it will be imperative to carefully define their mechanism of cellular entry. In fact, the mechanism of uptake of cationic amphiphilic gene carriers by cells is still a matter of debate. Early work suggested that cationic amphiphile-DNA complexes could enter the cell via fusion with the plasma membrane (1). Although attractive given its membranous nature, lipid mixing assays did not reveal a correlation between fusion events of lipoplexes with cellular membranes and their transfection efficiency (2-5). Next to fusion, a mechanism that involves internalization via endocytosis has received most support thus far (6 -8). The evidence is often based on the application of metabolic inhibitors of endocytosis like chloroquin, monensin, and NH 4 Cl. However, the precise effect of a certain metabolic inhibitor is often difficult to interpret because both attenuation and diminution of transfection efficiency have been reported, while using one and the same inhibitor (6, 9 -1...

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

“…To obtain further support for this notion, we next investigated whether correct targeting could be reestablished, after replenishment of cellular cholesterol by addition of CD/Chol complexes, as described previously (Zuhorn et al, 2002). As can be seen in Figure 9, F and G, after restoring cholesterol levels, MDR1-GFP was no longer detected at the BL membrane, displaying again its prominent presence at the BC surface, thus indicating that cholesterol is crucial for the correct AP trafficking of MDR1.…”

“…Replenishment of depleted cholesterol was carried out by incubating the cells with preformed cyclodextrin/cholesterol complexes as described previously (Zuhorn et al, 2002). Briefly, after cholesterol depletion, HepG2 cells were washed with serum-free medium and incubated 3 h at 37°C in the presence of CD-cholesterol (CD/Chol) complexes (400 g/ml cholesterol) diluted in serum-free medium and subsequently used for cholesterol determination or for MDR1-GFP distribution.…”

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

“…39 hTF is known to be taken up via CDE while LacCer is internalized via CIE where is associated with lipid rafts. 40 Fig. S1 and S2 † show the CDE and CIE inhibitors suppressing the hTF and LacCer uptake in SHEP and HepG2 cells, respectively.…”

Intracellular trafficking of nonviral vectors

“…While several drugs that can inhibit both clathrin-and caveolae-mediated pathways of cell entry reduce transfection by SAINT-2/DOPE liposomes, HepG2 cells lack caveolae, thus implicating a clathrinmediated route in these cells. 92 Transfection by these lipoplexes could be substantially reduced by cholesterol depletion with methyl-b-cyclodextrin (MbCD) or chlorpromazine, while an inhibitor of caveolae-mediated endocytosis, 93 filipin III, blocked transfection by 20%. Localization of the lipoplexes with Tf receptor, used as a marker for clathrin-coated endosomes, 94 and inhibition of internalization by dominant-negative Eps15, and K + depletion, which both arrest coated pit formation, confirmed a clathrin-mediated route of uptake.…”

“…Apparently, the size of the complex matters, as 500 nm complexes entered cells through noncoated vesicles, but not through coated vesicles, which have been reported to be 80-100 nm in diameter. 92 Importantly, complexes of this size, while able to enter cells, yielded relatively low levels of gene transfer and expression, suggesting that clathrincoated endocytosis is linked to endosomolytic ability.…”