CL-DNA Complexes Research Articles

Uptake and transfection efficiency of PEGylated cationic liposome–DNA complexes with and without RGD-tagging

Steric stabilization of cationic liposome–DNA (CL–DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL–DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL–DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL–DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL–DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL–DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.

Packing DNA with disc-shaped bicelles

Cationic lipid–DNA (CL–DNA) complexes can be used as non-viral vectors to improve transfection efficiency in gene therapy. It is greatly desirable to develop new ways of packing DNA in order to make breakthroughs on the development of highly efficient DNA delivery systems. Different from the widely-studied vesicle based CL–DNA complexes, we have developed a novel cationic bicelle–DNA complex system by mixing cationic bicelles with DNA to form alternating stacks of disc bilayered plates and DNA arrays with a repeat distance of 7.5 nm. SAXS and TEM studies revealed that the DNA molecules encapsulated between the disc bilayered plates form ordered arrays with a spacing of around 4–5 nm. The number of stacking layers can be easily tuned from just a few layers to more than one hundred layers by adjusting the doping percentage of the charged lipids. This CB–DNA complex is quite stable against increases in temperature above the chain-melting temperature of the long chain lipids. Due to the flexible size tunability (number of stacking layers) and the unique structure of the packing of the DNA arrays with 50–100 nm size disc bilayered plates, the CB–DNA complexes are promising as novel non-viral vectors for gene delivery.

Endosomal escape and transfection efficiency of PEGylated cationic liposome–DNA complexes prepared with an acid-labile PEG-lipid

Cationic liposome–DNA (CL–DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL–DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL–DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL–DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL–DNA complexes are stable at pH 7.4 for more than 24 h, but the PEG chains are cleaved at pH 5 within 1 h, leading to complex aggregation. The acid-labile HPEG2K-CL–DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL–DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome.

PH-Sensitive Pegylated Cationic Lipid-DNA Complexes for Gene Delivery: Transfection Efficiency and Live Cell Imaging Studies

Gene therapy is a promising new approach for medicine, and able to target a wide variety of diseases. [1]. Cationic liposome (CL)-DNA complexes (also known as lipoplexes) are desirable non-viral gene vectors because of low immunogenicity, ease of scale-up procedures, etc.. To be viable for in vivo applications, CL-DNA complexes need to be stable in circulation, which can be achieved by addition of poly-(ethylene glycol) (PEG)-lipids. Unfortunately, PEG-lipids interfere with complex-cell membrane interactions that are vital for particle attachment and uptake and can inhibit membrane fusion required for endosomal escape. The benefit of increased stability from PEG-lipids, thus, comes at the cost of a reduction in transfection efficiency (TE).To recover the resulting loss in TE, we have designed and synthesized a hydrolysable acid-labile PEG-lipid (HPEG-lipid, PEG MW 2000) which is stable at physiological pH, but is cleaved at low pH. The natural acidification process of endosomes will liberate the PEG from the lipoplexes. We have studied the colloidal stability and transfection efficiency of these HPEG-lipoplexes in mammalian cell cultures. The acid sensitivity and the colloidal stability were characterized by TLC and dynamic light scattering, respectively. The HPEG-lipid is stable at neutral pH for more than 24 h, but degrades completely within 1 h at pH 4, leading to particle aggregation. HPEG-lipoplexes show lower toxicity and enhanced TE in comparison to lipoplexes stabilized with pH-stable PEG-lipids. Live-cell images show that both pH-sensitive and pH-stable PEG-lipoplexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation. Thus, the increased TE of the HPEG-lipoplexes can be attributed to efficient endosomal escape, enabled by the novel HPEG-lipid. Funded by NIH-GM-59288 and NSF-DMR-1101900.[1] Ewert, K. K. et al, Topics Curr. Chem. 2010, 296, 191-226

Cationic liposome–nucleic acid complexes: liquid crystal phases with applications in gene therapy

Cationic liposome (CL) carriers of nucleic acids are studied primarily because of their applications in gene delivery and gene silencing with CL-DNA and CL-siRNA (short-interfering RNA) complexes, respectively, and their implications for ongoing clinical gene therapy trials worldwide. A series of synchrotron-based small-angle X-ray scattering studies, dating back to 1997, has revealed that CL-nucleic acid complexes spontaneously assemble into distinct novel liquid crystalline phases of matter. Significantly, transfection efficiency (TE; a measure of expression of an exogenous gene that is transferred into the cell by the lipid carrier) has been found to be dependent on the liquid crystalline structure of complexes, with lamellar complexes showing strong dependence on membrane charge density (σM) and non-lamellar complexes exhibiting TE behaviour independent of σM. The review describes our current understanding of the structures of different liquid crystalline CL-nucleic acid complexes including the recently described gyroid cubic phase of CL-siRNA complexes used in gene silencing. It further makes apparent that the long-term goal of developing optimised liquid crystalline CL-nucleic acid complexes for successful medical applications requires a comprehensive understanding of the nature of the interactions of distinctly structured complexes with cell membranes and events leading to release of active nucleic acids within the cell cytoplasm.

Cationic Lipid Vectors for Gene Delivery: Distinct Pathways of Endosomal Release

Cationic liposomes (CLs) are used as non-viral gene vectors in worldwide human clinical trials of gene therapy. Because our understanding of the mechanisms of action of CL-DNA complexes remains poor and transfection efficiencies remain low compared to gene delivery with viral vectors, significant additional insights and discoveries will be required before the development of efficient chemical carriers suitable for long-term therapeutic applications. (For example, virtually all current human gene therapy protocols using lipofection as a vector contain cholesterol even though very little is understood about how these cholesterol-containing complexes improve transfection in-vivo.) Our studies on CL-DNA complexes employs synchrotron x-ray diffraction to reveal structure, confocal microscopy to reveal CL-DNA pathways and interactions with cells, and transfection efficiency measurements. The combined data indicate that the mechanism of gene release from complexes in the cell cytoplasm is dependent on their precise liquid crystalline structural nature and the physical and chemical parameters (e.g., the membrane charge density, membrane composition) of the complexes. The talk will describe results on cationic complexes with and without cholesterol emphasizing the differences in the interactions between the membranes of complexes with endosomal membranes leading to fusion and release into the cytoplasm. Funding provided by NIH GM-59288.

Effect of Nucleic Acid Length and Chemistry on Structure-Function Properties of Cationic Lipid-Nucleic Acid Complexes

The success of gene therapeutics is tied to the efficiency of transfer of the nucleic acid (NA) into a target cell type with minimal side effects. Synthetic vectors for delivery, such as cationic lipids (CL), can satisfy these criteria but only though a fundamental understanding of the structure-function relationship between CL-NA. The delivery efficiency of CL-DNA complexes follows a universal relationship that depends on the membrane charge density of the lipid layers but is independent of the type of CL. Our work on CL-siRNA complexes reveals stark differences in delivery behavior: the efficiency is not only broadly lower when compared with equivalent composition CL-DNA but also depends on the valence of the CL with multivalent favored over monovalent [1]. X-ray structural work indicates that siRNA ordering in CL-siRNA is isotropic, contrasting the smectic ordering of DNA in CL-DNA. We hypothesize that this isotropic behavior prevents optimal packing of the siRNA and is responsible for lower efficiencies. This behavior is in general agreement with Onsager rigid rod criteria for liquid crystal order transitions that depend on the dimensional anisotropy of the molecules. Our work with analogues CL-short DNA (sDNA) contradicts these findings; ordered sDNA phases can be seen in the 2D environment between the lipid layers well below the Onsager limit. This is consistent with recent work on sDNA suspended in bulk water [2]. While the ordered phase transitions of sDNA depend on length they also depend on the chemistry of the NA ends. These considerations may be applied to the siRNA in an attempt to improve the CL-siRNA packing efficiency.Supported by NIH GM-59288, DOE DE-FG02-06ER46314, NSF DMR-0803103[1] Bouxsein et al. Biochemistry (2007) 46, 4785.[2] Nakata et al. Science (2007) 318, 5854.

Liquid Crystalline Phases of Dendritic Lipid−DNA Self-Assemblies: Lamellar, Hexagonal, and DNA Bundles

The prospects of gene therapy have generated unprecedented interest in the properties and structures of complexes of nucleic acids (NAs) with cationic liposomes (CLs), which are used as nonviral NA carriers in worldwide clinical trials. An improved understanding of the mechanisms of action of CL-NA complexes is required to enable their widespread therapeutic use. In prior studies of CL-mediated DNA delivery, membrane charge density (sigma(M)) was identified as a key parameter for transfection efficiency (TE) of lamellar (L(alpha)(C)) CL-DNA complexes. The TE of CL-DNA complexes containing cationic lipids with headgroup valencies from 1+ to 5+ follows a universal bell-shaped curve as a function of sigma(M). As we report here, the TE of CL-DNA complexes containing new multivalent lipids with dendritic headgroups (DLs) strongly deviates from this curve at high sigma(M). We have investigated four DLs, MVLG2 (4+), MVLG3 (8+), MVLBisG1 (8+), and MVLBisG2 (16+), in mixtures with neutral 1,2-dioleoyl-sn-glycerophosphatidyl-choline (DOPC). To understand the TE behavior, we have performed X-ray diffraction (XRD), optical microscopy, and cryo-TEM studies of the DL/DOPC mixtures and their DNA complexes. XRD reveals a complex phase behavior of DL-DNA complexes which strongly depends on the headgroup charge. MVLG2(4+)/DOPC-DNA complexes exhibit the lamellar phase at all molar fractions of DL, Phi(DL). In stark contrast, MVLBisG2(16+)/ DOPC-DNA complexes remain lamellar only for Phi(DL) < or = 0.2. In a narrow regime around Phi(DL) = 0.25, the hexagonal phase H(I)(C), consisting of a hexagonal lattice of cylindrical lipid micelles and a DNA honeycomb lattice, is formed. At Phi(DL) > 0.3, XRD suggests formation of a distorted H(I)(C) phase. For Phi(DL) > or = 0.5 under high salt conditions, this phase coexists with a bundle phase of DNA condensed by the depletion-attraction effect of DL micelles. The transitions at high sigma(M) from the lamellar phase to the new hexagonal phases of DL-DNA complexes coincide with the deviation from the universal TE behavior of lamellar complexes. The observed high TE, which is independent of sigma(M), strongly suggests a novel mechanism of action for these DL-DNA complex phases.

Transitions between Distinct Compaction Regimes in Complexes of Multivalent Cationic Lipids and DNA

Cationic lipids (CLs) have found widespread use as nonviral gene carriers (vectors), including applications in clinical trials of gene therapy. However, their observed transfection efficiencies (TEs) are inferior to those of viral vectors, providing a strong incentive for a detailed understanding of CL-DNA complex behavior. In recent systematic studies employing monovalent as well as newly synthesized multivalent lipids (MVLs), the membrane cationic charge density has been identified as a key parameter governing the TE of lamellar CL-DNA complexes. In this work, we use x-ray scattering and molecular simulations to investigate the structural properties of complexes containing MVLs. At low mole fraction of neutral lipids (NLs), Φ NL, the complexes show dramatic DNA compaction, down to essentially close-packed DNA arrays with a DNA interaxial spacing d DNA = 25 Å. A gradual increase in Φ NL does not lead to a continuous increase in d DNA as observed for DNA complexes of monovalent CLs. Instead, distinct spacing regimes exist, with sharp transitions between the regimes. Three packing states have been identified: 1), close packed, 2), condensed, but not close packed, with d DNA = 27–28 Å, and 3), an expanded state, where d DNA increases gradually with Φ NL. Based on our experimental and computational results, we conclude that the DNA condensation is mediated by the multivalent cationic lipids, which assemble between the negatively charged DNA rods. Quite remarkably, the computational results show that the less tightly packed structure in regime 2 is thermodynamically more stable than the close packed structure in regime 1. Accordingly, the constant DNA spacing observed in regime 2 is attributed to lateral phase coexistence between this stable CL-DNA complex and neutral membranes. This finding may explain the reduced TE measured for such complexes: transfection involves endosomal escape and disassembly of the complex, and these processes are inhibited by the high thermodynamic stability. Our results, which demonstrate the existence of an inverse correlation between the stability and transfection activity of lamellar CL-DNA complexes are, therefore, consistent with a recently proposed model of cellular entry.

Tumor specific ultrasound enhanced gene transfer in vivo with novel liposomal bubbles

Bubble liposomes (liposomes which entrap an ultrasound imaging gas) may constitute a unique system for delivering various molecules efficiently into mammalian cells in vitro. In this study, Bubble liposomes were compared with cationic lipid (CL)–DNA complexes as potential gene delivery carriers into tumor in vivo. The delivery of genes by Bubble liposomes depended on the intensity of the applied ultrasound. Transfection efficiency plateaued at 0.7 W/cm 2 ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, Bubble liposomes could introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells and solid tumor tissue. We conclude that the combination of Bubble liposomes and ultrasound is a minimally-invasive and tumor specific gene transfer method in vivo.

Structure and Gene Silencing Activities of Monovalent and Pentavalent Cationic Lipid Vectors Complexed with siRNA

Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (rhochg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given rhochg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and rhochg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.

Structural thermodynamics of lamellar cationic lipid-DNA complex: DNA compressibility modulus

We have studied theoretically the compressibility modulus B of DNA and complexation adsorption isotherms of DNA and lipids, as a function of DNA spacing d(DNA) and NaCl electrolyte concentration, respectively, in isoelectric states of lamellar DNA/cationic lipid (CL) self-assemblies. The electrostatic free energy derived from the Poisson-Boltzmann theory predicts partial agreement with measured B values for interhelical separations d(DNA)>33 A when use is made of a fit of hydration repulsion from bulk DNA hexagonal phases in solution. For lower interchain separations the prediction worsens due to the hydration interaction that overcomes the electrostatic contribution. An exact match of the system's counterion electrochemical potentials and the coions of salt in aqueous phase leads to the electrostatic part of the free energy that renders isotherms of d(DNA) versus ionic strength in qualitative consistency with general trends of available experimental data of CL-DNA complexes.

Cationic lipid-DNA complexes for gene therapy: understanding the relationship between complex structure and gene delivery pathways at the molecular level.

Cationic liposomes (CLs) are used as gene vectors (carriers) in worldwide human clinical trials of non-viral gene therapy. These lipid-gene complexes have the potential of transferring large pieces of DNA of up to 1 million base-pairs into cells. As our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies are still low when compared to gene delivery with viral vectors. We describe recent studies with a combination of techniques (synchrotron x-ray diffraction for structure determination, laser-scanning confocal microscopy to probe the interactions of CL-DNA particles with cells, and luciferase reporter-gene expression assays to measure transfection efficiencies in mammalian cells), which collectively are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their transfection efficiency. The work described here is applicable to transfection optimization in ex vivo cell transfection, where cells are removed and returned to patients after transfection. CL-DNA complexes primarily form a multilayered sandwich structure with DNA layered between the cationic lipids (labeled L(alpha)(C)). On rare occasions, an inverted hexagonal structure with DNA encapsulated in lipid tubules (labeled H(II)(C)) is observed. A major recent insight is that for L(alpha)(C) complexes the membrane charge density sigma(M) of the CL-vector, rather than the charge of the cationic lipid alone, is a key universal parameter that governs the transfection efficiency of L(alpha)(C) complexes in cells. The parameter sigma(M) is a measure of the average charge per unit area of the membrane, thus taking into account the amount of neutral lipids. In contrast to L(alpha)(C) complexes, H(II)(C) complexes containing the lipid 1,2-dioleoyl-sn-glycerophosphatidylethanolamine (DOPE) exhibit no dependence on sigma(M). The current limiting factor to transfection by cationic lipid vectors appears to be the tight association of a fraction of the delivered exogenous DNA with cationic cellular molecules, which may prevent optimal transcriptional activity. Future directions are outlined, which make use of surface-functionalized CL-DNA complexes suitable for transfection in vivo.

Three-Dimensional Imaging of Lipid Gene-Carriers: Membrane Charge Density Controls Universal Transfection Behavior in Lamellar Cationic Liposome-DNA Complexes

Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L(alpha)(C) and inverted hexagonal H(II)(C) nanostructures, and mouse fibroblast cells. Confocal images of L(alpha)(C) complexes in cells identified two regimes. For low membrane charge density (sigma(M)), DNA remained trapped in CL-vectors. By contrast, for high sigma(M), released DNA was observed in the cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L(alpha)(C)-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of sigma(M), identifying it as a key universal parameter. The universal curve for transfection by L(alpha)(C) complexes climbs exponentially over approximately four decades with increasing sigma(M) below an optimal charge density (sigma(M)(*)), and saturates for at a value rivaling the high transfection efficiency of H(II)(C) complexes. In contrast, the transfection efficiency of H(II)(C) complexes is independent of sigma(M). The exponential dependence of TE on sigma(M) for L(alpha)(C) complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sigma(M) lowers the barrier. In the saturated TE regime, for both L(alpha)(C) complexes and H(II)(C), confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.

Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery.

Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. This paper describes the efficient and facile synthesis and the characterization of a new multivalent cationic lipid with a double-branched headgroup structure for gene delivery applications. The synthetic scheme can be extended to give cationic lipids of different charge, spacer, or lipid chain length. The chemical and physical properties of self-assembled complexes of the cationic liposomes (CLs) with DNA give indications of why multivalent cationic lipids possess superior transfection properties. The lipid bears a headgroup with five charges in the fully protonated state, which is attached to an unsaturated double-chain hydrophobic moiety based on 3,4-dihydroxybenzoic acid. Liposomes consisting of the new multivalent lipid and the neutral lipid 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) were used to prepare complexes with DNA. Investigations of the structures of these complexes by optical microscopy and small-angle X-ray scattering reveal a lamellar L(alpha)(C) phase of CL-DNA complexes with the DNA molecules sandwiched between bilayers of the lipids. Experiments using plasmid DNA containing the firefly luciferase reporter gene show that these complexes efficiently transfect mammalian cells. When compared to the monovalent cationic lipid 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP), the higher charge density of the membranes of CL-DNA complexes achievable with the new multivalent lipid greatly increases transfection efficiency in the regime of small molar ratios of cationic to neutral lipid. This is desired to minimize the known toxicity effects of cationic lipids.

Structure and Structure—Function Studies of Lipid/Plasmid DNA Complexes

Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear λ-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled LCα with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled HCII) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, ΦDOPC = 0.72) and a low transfectant complex (DOTAP/DOPC, ΦDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.

Lipid-DNA Complex Formation: Reorganization and Rupture of Lipid Vesicles in the Presence of DNA As Observed by Cryoelectron Microscopy

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular “glue” enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a “template” vesicle or a lipid-DNA complex.

DNA at membrane surfaces: An experimental overview

Atomic force microscopy studies of DNA attached to rigid surfaces were initially motivated by the development-of methods which would stretch DNA for the purposes of rapid sequencing. Currently there is much interest in studies of multilayers of DNA chains self-assembled on membranes which form spontaneously when DNA adsorbs onto oppositely charged cationic liposomes (CLs). A major motivation for elucidating the structures and interactions in these CL-DNA complexes arises for two reasons. The first is that they are known to mimic certain characteristics of viruses by being efficient chemical carriers of genes (DNA sections) for delivery in cells. The second is that they are models of studies of DNA condensation phases in two dimensions. DNA-membrane interactions should also provide clues for the relevant molecular forces in the condensation of DNA in chromosomes and viral capsids. The particular complexes described in this review contain linear DNA forming a new ‘hybrid’ phase of matter; that is, the DNA chains form a finite size two dimensional smectic coupled to the three dimensional smectic phase of membranes.