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The cytosol of mammalian cells is a crowded environment containing soluble proteins and a network of cytoskeletal filaments. Gene delivery by synthetic vectors involves the endocytosis of DNA-polycation complexes, escape from endosomes, and diffusion of non-complexed DNA through the cytosol to reach the nucleus. We found previously that the translational diffusion of large DNAs (>250 bp) in cytoplasm was greatly slowed compared with that of smaller DNAs (Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. (2000) J. Biol. Chem. 275, 1625-1629). To determine the mechanisms responsible for size-dependent DNA diffusion, we used fluorescence correlation spectroscopy to measure the diffusion of single fluorophore-labeled DNAs in crowded solutions, cytosol extracts, actin network, and living cells. DNA diffusion (D) in solutions made crowded with Ficoll-70 (up to 40 weight percentage) or soluble cytosol extracts (up to 100 mg/ml) relative to diffusion of the same sized DNAs in saline (D/D(o)) was approximately independent of DNA size (20-4500 bp), quite different from the strong reduction in D/D(o) in the cytoplasm of living cells. However, the reduced D/D(o) with increasing DNA size was closely reproduced in solutions containing cross-linked actin filaments assembled with gelsolin, whereas soluble macromolecules of the same size and concentration did not reduce D/D(o). In intact cells microinjected with fluorescent DNAs and studied by fluorescence correlation spectroscopy or photobleaching methods, D/D(o) was reduced by 5-150-fold (20-6000 bp); however, the size-dependent reduction in D/D(o) was abolished after actin cytoskeleton disruption. Our results identify the actin cytoskeleton as a major barrier restricting cytoplasmic transport of non-complexed DNA in non-viral gene transfer.

Aqueous environments in living cells are crowded, with up to >50 wt% small and macromolecule-size solutes. We investigated quantitatively one important consequence of molecular crowding-reduced diffusion of biologically important solutes. Fluorescence correlation spectroscopy (FCS) was used to measure the diffusion of a series of fluorescent small solutes and macromolecules. In water, diffusion coefficients (D o w ) were (in cm 2 /s × 10 - 8 ): rhodamine green (270), albumin (52), dextrans (75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5 kb) and polystyrene nanospheres (5.4, 20 nm diameter; 2.3, 100 nm). Aqueous-phase diffusion (D w ) in solutions crowded with Ficoll-70 (0-60 wt%) was reduced by up to 650-fold in an exponential manner: D w =D o w exp(-[C]/[C] e x p ), where [C] e x p is the concentration (in wt%) of crowding agent reducing D o w by 63%. FCS data for all solutes and Ficoll-70 concentrations fitted well to a model of single-component, simple (non-anomalous) diffusion. Interestingly [C] e x p were nearly identical (11 ′ 2 wt%, SD) for diffusion of the very different types of macromolecules in Ficoll-70 solutions. However, [C] e x p was dependent on the nature of the crowding agent: for example, [C] e x p for diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500kDa dextran. Our results indicate that molecular crowding can greatly reduce aqueous-phase diffusion of biologically important macromolecules, and demonstrate a previously unrecognized insensitivity of crowding effects on the size and characteristics of the diffusing species.

Controlling the size of condensed DNA particles is a key determinant for their diffusion in vivo as well as for gene delivery to target cells. Towards this goal, DNA molecules were compacted individually by cationic thiol-detergents into discrete nanometric entities. These particles were then stabilized by air-induced dimerization of the detergent into a disulfide lipid on the template DNA. Using a tetradecane-cysteine-ornithine (C 14 COrn) detergent, a solution of 5.5 Kb plasmid DNA was thus converted into a monodisperse population of 35-nm particles. The stability of the complexes, as well as their size, morphology and transfection efficiencies were investigated. Surprisingly, the electrophoretic mobility of the quasi-neutral condensed DNA was found higher than that of the extended DNA polyanion. The diameters of particles resulting from the condensation of DNA of various sizes was measured by dynamic light scattering and found to vary as the cubic root of the DNA size. In an attempt to extend their biodistribution and to target tumour cells, we have prepared folate-poly(ethylene oxide)-coated particles that were shown to bind to the cell-surface folate receptor.

The stability of the complexes between DNA and nonviral vectors is a crucial parameter for efficient gene delivery into target cells. The stability must be high enough to prevent any dissociation during interaction with the plasma membrane but low enough to allow the dissociation that is required for efficient internalization into the nucleus. In this report, we investigated the stability of complexes of DNA with two cysteine surfactants (guanidinocysteine N-decylamide, C10−CG+, and ornithinyl-cysteinyl-tetradecylamide, C14−CO), able to convert themselves, via oxidative dimerization, into cationic cystine lipids. To this end, we determined the critical aggregation concentration (cac) and the binding constants of the surfactants for DNA by using the fluorescence quenching of the DNA bis-intercalating agent, YOYO-1, that results from the dye clustering induced by the collapse of DNA. The cac's of C10−CG+ and C14−CO monomeric forms are 2.5 and 1 μM, respectively, and are slightly less than the 5 μM value for...

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.

Intracellular transport of DNA carriers is a fundamental step of gene delivery. By combining both theoretical and numerical approaches we study here single and several viruses and DNA particles trafficking in the cell cytoplasm to a small nuclear pore. We present a physical model to account for certain aspects of cellular organization, starting with the observation that a viral trajectory consists of epochs of pure diffusion and epochs of active transport along microtubules. We define a general degradation rate to describe the limitations of the delivery of plasmid or viral particles to a nuclear pore imposed by various types of direct and indirect hydrolysis activity inside the cytoplasm. By replacing the switching dynamics by a single steady state stochastic description, we obtain estimates for the probability and the mean time for the first one of many particles to go from the cell membrane to a small nuclear pore. Computational simulations confirm that our model can be used to analyze and interpret viral trajectories and estimate quantitatively the success of nuclear delivery.

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.

Nucleic acids delivery vectors have shown promising therapeutic potential in model systems. However, comparable clinical success is delayed essentially because of their poor biodistribution and of their ineffective intracellular trafficking. The size of condensed DNA particles is a key determinant for in vivo diffusion, as well as for gene delivery to the cell nucleus. Towards this goal, we have developed cationic thiol-detergents that individually compact plasmid DNA molecules into anionic particles. These particles are then 'stabilized' by air-induced dimerization of the detergent into a disulfide lipid on the template DNA. The particles all measure approximately 30 nm, which corresponds to the volume of a single molecule of plasmid DNA. The gel electrophoretic mobility of the anionic particles was found to be higher than that of the plasmid DNA itself. Similarly, particles formed with a 31-mer oligonucleotide measured 19 nm. Improved in vivo diffusion, as well as improved intracellular trafficking may be inferred from the faster migration of the complexes. Moreover, the size of the particles remains compatible with nuclear pore crossing. Finally, in an attempt to improve the biodistribution of these particles, we have coated the monomolecular particles with a poly(ethylene glycol) corona.

Gene delivery is too complex to be performed with a single carrier molecule. Synthetic multicomponent vectors are being designed that mimic key properties of viruses. Some solutions, such as diverting cell-anchoring molecules or the endogenous nuclear import machinery from their normal function, are directly copied from bacteria and viruses. Some other solutions are original ones: monomolecular genome condensation via detergent dimerization or endosome disruption by the proton sponge effect are not exploited by natural cell invaders. All these components, however, still have to be assembled into a unique supramolecular system, an 'artificial virus'.

International audience; Cationic amphiphile-mediated delivery of plasmid DNA is the non-viral gene transfer method most often used. In the present work, we considered a new cysteine-detergent, ornithinyl-cysteinyl-tetradecylamide (C(14)-CO), able to convert itself, via oxidative dimerization, into a cationic cystine-lipid. By using fluorescence techniques, we first characterized the structure of complexes of plasmid DNA with C(14)-CO molecules either kept as monomers, or oxidized into dimers. Both forms are able to condense DNA, with the formation of hydrophobic micelle-like domains along the DNA chain. Domains with a larger molecular order were obtained with dimeric C(14)-CO/DNA complexes. In a second step, the interactions of these complexes with lipid vesicles considered as membrane models were investigated. In the presence of vesicles, we observed a decondensation of the DNA involved in complexes obtained with C(14)-CO monomers. With anionic vesicles, the DNA is released into the bulk solution, while with neutral vesicles, it remains bound to the vesicles via electrostatic interactions with inserted C(14)-CO molecules. In sharp contrast, the complexes with C(14)-CO dimers are unaffected by the addition of either neutral or anionic vesicles and show no interaction with them. These results may partly explain the low transfection efficiency of these complexes at the +/-charge ratios used in this study.

Gene delivery by synthetic vectors is a multi-step process that relies on the endocytosis of the DNA particles, escape from endosomes, and diffusion of the DNA through the cytosol before the import in the nucleus. The cytoplasm of mammalian cells is a complex environment containing a fluid phase, a high concentration of soluble proteins, and a network of cytoskeletal filaments. We found previously that the translational diffusion of small DNAs ( 250 bp) were greatly slowed (Dcyto/Dw/Dw< 0.05) (J. Biol. Chem. 275:1625–1629, 2000). To investigate the mechanisms responsible for size-dependent DNA diffusion, we measured by fluorescence correlation spectroscopy the diffusion of fluorescent linear, double-stranded DNAs in saline and artificial crowded solutions, cytosol extracts, actin network and cell cytoplasm. DNA fragments containing a single rhodamine green molecule were generated by PCR. In saline solution, DNA diffusion coefficients decreased with DNA size according to: Dw = 8.1 × 10−6cm2/s × (base pair) −0.71. In solutions containing the inert crowding agent Ficoll-70, DNA mobility was reduced exponentially with the Ficoll-70 concentration (5-fold at 18 wt% Ficoll-70); however, neither Ficoll-70 nor concentrated cytosolic soluble fractions reproduced size-dependent reduction in Dcyto/Dw as found in intact cells. In contrast, reduced Dcyto/Dw with increasing DNA size was reproduced in saline solutions crowded with actin filaments, the principal component of the cytoskeleton. Compact globular macromolecules of same size were not slowed, implicating the extended conformation of the DNA in its impaired diffusion. In intact cells microinjected with the fluorescent DNAs, Dcyto/Dw was greatly reduced as found in prior photobleaching measurements; however, the size-dependent reduction in Dcyto/Dw was eliminated with cytoskeletal disrupters (cytochalasin D, latrunculin B) and was dependent on cell volume. Our results implicate actin cytoskeleton as the principal determinant of the restricted DNA mobility in cytoplasm, thus identifying a new barrier in restricting DNA movement to the nucleus.

International audience; Cationic lipids are efficient vectors for DNA delivery in vitro. However, they condense DNA into large polymorphic particles, which severely limits their in vivo performances due to size-restricted diffusion. In contrast, detergents are capable of collapsing DNA into smaller particles but do not mediate cell transfection per se. We have succeeded in retaining the interesting features of both types of amphiphiles in a two-step process leading to monomolecular DNA particles stable in physiologic medium. Anionic DNA molecules were first individually condensed with a designed cationic cysteine-based detergent. The resulting small particles were then stabilised by spontaneous thiol dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. Laser light scattering as well as electron microscopy revealed a monodisperse population of spherical particles that were stable for days in physiological conditions. With an appropriate choice of hydrocarbon chain length, monomolecular complexes exhibiting a typical lipid/DNA internal structure could be obtained. Their in vitro cell transfection properties compare favourably with those of Lipofectamine and PEI.