Synthesis of Pyridinium Amphiphiles Used for Transfection and Some Characteristics of Amphiphile/DNA Complex Formation

Meekel, A. A. P.; Wagenaar, Anno; Šmisterová, J.; Kroeze, Jessica E.; Haadsma, Peter; Bosgraaf, Bouke; Stuart, Marc C. A.; Brisson, Alain; Ruiters, Marcel H. J.; Hoekstra, Dick; Engberts, Jan B. F. N.

doi:10.1002/(sici)1099-0690(200002)2000:4<665::aid-ejoc665>3.0.co;2-a

Cited by 70 publications

(65 citation statements)

References 19 publications

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“…In short, the superior transfection properties of SAINT-2 lipoplexes, as described in previous work (12,15) appears to be related to its ability to undergo a lamellar to a nonlamellar phase transition, which requires the presence of DOPE and which is promoted by charge neutralizing conditions as provided at physiologically relevant conditions. Specifically, the transition from the lamellar to the hexagonal organization of SAINT-2-based lipoplexes enhances the cellular transfection efficiency 2-5-fold, with the net increment depending on the presence of DOPE and physiological salt conditions.…”

Section: Discussionsupporting

confidence: 67%

“…The ability of SAINT-2, compared with the SAINT-21 and -27 derivatives, to readily undergo a lamellar to nonlamellar organization and thereby strongly promote cationic lipid-mediated transfection can be rationalized by taking into account the molecular shape concept (12,33). Thus SAINT-2 shows the largest imbalance between the cross-sectional areas occupied by the hydrophobic and hydrophilic domains of its structure (Fig.…”

Section: Discussionmentioning

confidence: 63%

“…Note that cryo-TEM images of SAINT-2/DOPE lipoplexes prepared in water display an entirely different morphology, showing a lamellar structure similar to that of SAINT-27 and SAINT-21 lipoplexes (not shown; Fig. 5), as reported previously (12). To further investigate the structural organization underlying the morphological features of these various complexes, SAXS measurements were subsequently carried out.…”

Section: Resultsmentioning

confidence: 99%

“…The purpose of the current study was to obtain such insight by using one particular class of cationic pyridinium-derived lipids, abbreviated as SAINTs (12,15,32). The effect of the overall shape of the amphiphiles on the colloidal stability of the bilayers formed with DOPE (1:1) and the impact of this stability/instability on the polymorphism of SAINT/DOPE-DNA complexes within a group of structurally highly related compounds, although differing in their ability to deliver DNA into COS-7 cells, was investigated by electron microscopy, by small angle x-ray scattering, and in transfection studies.…”

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confidence: 99%

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Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid/Dioleylphosphatidylethanolamine-DNA Complexes and the Efficiency of Gene Delivery

Šmisterová

Wagenaar

Stuart

et al. 2001

Journal of Biological Chemistry

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174

144

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Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mM NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid/Dioleylphosphatidylethanolamine-DNA Complexes and the Efficiency of Gene Delivery

Šmisterová

Wagenaar

Stuart

et al. 2001

Journal of Biological Chemistry

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174

144

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“…An equal volume of a 2 g/ml plasmid DNA solution (pEGFP-N1, CLONTECH) was added to a 30 M solution of SAINT-2/DOPE (1:1) liposomes, resulting in the formation of lipoplexes with a molar charge ratio of 2.5:1 (cationic lipid:DNA). SAINT-2 was synthesized as described in detail elsewhere (24); DOPE was purchased from Avanti Polar Lipids (Birmingham, AL). One day before transfection, COS7 cells were seeded into 6-wells plates at 3 ϫ 10 5 cells per well.…”

Section: Methodsmentioning

confidence: 99%

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

Zuhorn¹,

Kalicharan²,

Hoekstra³

2002

Journal of Biological Chemistry

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298

219

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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...

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Synthesis and Characteristics of Biodegradable Pyridinium Amphiphiles Used for in vitro DNA Delivery

Roosjen

Šmisterová²,

Driessen³

et al. 2002

Eur. J. Org. Chem.

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Pyridinium amphiphiles have found practical application for the delivery of DNA into eukaryotic cells. A general synthetic method starting from (iso)nicotinoyl chloride has been devised for the preparation of pyridinium amphiphiles based on (bio)degradable esters, allowing structural variation both in the hydrophobic part and in the headgroup area. By means of differential scanning calorimetry, transmission electron mi-

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Synthesis of Pyridinium Amphiphiles Used for Transfection and Some Characteristics of Amphiphile/DNA Complex Formation

Cited by 70 publications

References 19 publications

Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid/Dioleylphosphatidylethanolamine-DNA Complexes and the Efficiency of Gene Delivery

Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid/Dioleylphosphatidylethanolamine-DNA Complexes and the Efficiency of Gene Delivery

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

Synthesis and Characteristics of Biodegradable Pyridinium Amphiphiles Used for in vitro DNA Delivery

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