lipid-DNA Complexes Research Articles

Efficacy of cationic lipid–DNA complexes (CLDC) on hepatitis B virus in transgenic mice

Cationic lipid–DNA (non-coding) complexes (CLDC) are activators of the innate immune response that increase survival of rodents with some acute viral infections and cancers. CLDC were evaluated for their ability to impact viral DNA levels in transgenic mice carrying an infectious clone of hepatitis B virus (HBV). Mice used in the studies were diet-restricted as nursing pups from solid food, because the expression of HBV DNA in the liver was increased above background levels in some mice with this restriction. Survival surgery was performed on these mice to obtain liver biopsies from which to select animals with suitable levels of liver HBV DNA for entry into the experimental protocols. Intravenous administration of 5 μg/mouse of CLDC on days 1, 7 and 13 reduced liver HBV DNA to similar low levels achieved with the positive control, adefovir dipivoxil. In a subsequent experiment, the same treatment schedule was used to determine that the minimal effective CLDC dose was between 0.5 and 0.05 μg/mouse. Selective cytokines were increased in the livers of CLDC-treated compared to placebo-treated mice in a dose-responsive manner. CLDC were effective in reducing liver HBV DNA and could be considered for further evaluation in other hepatitis models.

Potent Anti-Leukemic Activity of a Cationic Lipid-DNA Complex.

Cationic Lipid and DNA Complex (CLDC) is a chemically defined preparation of lipids and non-coding bacterial plasmid DNA able to activate antigen presenting and immune effector cells and thereby enhance cellular immune responses. We tested this preparation subcutaneously (SC) in two models of transplantable murine leukemia: 32D -(bcr-abl)p210 (leukemogenic in C3H/HEJ animals) and WEHI-3B cells (leukemogenic in BALB-C mice.) In multiple experiments using the 32Dp210 model a single SC dose of CLDC (200 ul) prevented or significantly delayed death from leukemia when delivered between -1 and 16 days from leukemic challenge. Specifically, control animals died at 28 days (SD=2.73) of leukemia whereas 20/24 similarly leukemia challenged but CLDC treated animals were alive >71 days (p<0.0001 − 4/20 animals died of leukemia). Animals dying of leukemia had swollen spleens and leukemic infiltration of spleen and liver on histopathological evaluation. Similarly Balb-C animals challenged with WEHI-3B cells died at a median of 30 days unless treated with CLDC (1 dose day 6 − 4/5 alive >day 40 p=0.0017). A green fluorescence protein (GFP) expressing subclone of 32Dp210 allowed quantitation of leukemia in peripheral blood (PB). PB leukocytes were 20% GFP positive on day 19. After CLDC on day 19, day 21 PB showed only 2% GFP. Animals receiving 32D-p210-GFP and treated with CLDC and CD8 antibody on day 19 showed higher GFP in followup and died earlier (median day 35) than animals treated with CLDC alone (>42 days) but later than animals receiving no treatment after leukemic challenge (median 23 days, p=0.0002.) See Figure below. We conclude that CLDC shows potent anti-leukemia activity in two murine models of AML. Antibody experiments suggest that CD8 positive effector cells contribute to this effect. Additional experiments are maturing and will be reported. This preparation has promise for activity in human acute leukemias. [Display omitted]

Effect of the Hydrocarbon Chain and Polymethylene Spacer Lengths on Gene Transfection Efficacies of Gemini Lipids Based on Aromatic Backbone

Design, syntheses, and gene delivery efficacies of fifteen novel gemini (dimeric) and three monomeric cationic lipids anchored on an aromatic backbone have been described. Each new lipid has been used for liposome formation, and optimal formulations were used to determine the structure-activity correlation of the gene transfection efficacies of these lipids in HeLa and HT1080 cells. The results of the present investigation bring out the effect of hydrocarbon chain lengths and the length of the spacer between the headgroups on gene transfection efficiencies of the cationic gemini lipids based on aromatic backbone. The lipids bearing n-C 14H 29 hydrocarbon chain lengths have been found to be the best transfecting agents compared to their counterparts with n-C 16H 33 and n-C 12H 25 chains in HeLa cells. On the other hand, in HT1080 cells, the lipids based on n-C 12H 25 and n-C 14H 29 chains were found to be more potent transfecting agents than lipids possessing n-C 16H 33 chains. Transmission electron microscopy examination revealed the existence of spherical lipid-DNA complexes.

Structural and functional diversity of artificial membranes based on cationic lipodipeptides

Membrane-forming properties of cationic amphiphiles based on lipodipeptides were studied with special reference to resistance of amphiphilic dispersions (pure amphiphil and amphiphil/cholesterol mixtures) to a destabilizing factor (ethanol). Values of critical micelle concentrations and phase transition temperatures were determined and microphotographs of lipid dispersions and lipid-DNA complexes in aqueous environment were obtained.

The role of actin and microtubule networks in plasmid DNA intracellular trafficking.

This work is focused on the function of the microtubule and actin networks in plasmid DNA transport during liposomal transfection. We observed strong binding of plasmid DNA-lipid complexes (lipoplexes) to both networks and directional long-range motion of these lipoplexes along the microtubules. Disruption of either of these networks led to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and accumulation of plasmid DNA in large aggregates at the cell periphery. Our findings show an indispensable but different role of both types of cytoskeleton, actin and microtubular, in the processes of gene delivery.

Computational and Analytical Modeling of Cationic Lipid-DNA Complexes

We present a theoretical study of the physical properties of cationic lipid-DNA (CL-DNA) complexes—a promising synthetically based nonviral carrier of DNA for gene therapy. The study is based on a coarse-grained molecular model, which is used in Monte Carlo simulations of mesoscopically large systems over timescales long enough to address experimental reality. In the present work, we focus on the statistical-mechanical behavior of lamellar complexes, which in Monte Carlo simulations self-assemble spontaneously from a disordered random initial state. We measure the DNA-interaxial spacing, d DNA, and the local cationic area charge density, σ M, for a wide range of values of the parameter ϕ c representing the fraction of cationic lipids. For weakly charged complexes (low values of ϕ c), we find that d DNA has a linear dependence on ϕ c − 1 , which is in excellent agreement with x-ray diffraction experimental data. We also observe, in qualitative agreement with previous Poisson-Boltzmann calculations of the system, large fluctuations in the local area charge density with a pronounced minimum of σ M halfway between adjacent DNA molecules. For highly-charged complexes (large ϕ c), we find moderate charge density fluctuations and observe deviations from linear dependence of d DNA on ϕ c − 1 . This last result, together with other findings such as the decrease in the effective stretching modulus of the complex and the increased rate at which pores are formed in the complex membranes, are indicative of the gradual loss of mechanical stability of the complex, which occurs when ϕ c becomes large. We suggest that this may be the origin of the recently observed enhanced transfection efficiency of lamellar CL-DNA complexes at high charge densities, because the completion of the transfection process requires the disassembly of the complex and the release of the DNA into the cytoplasm. Some of the structural properties of the system are also predicted by a continuum free energy minimization. The analysis, which semiquantitatively agrees with the computational results, shows that that mesoscale physical behavior of CL-DNA complexes is governed by interplay among electrostatic, elastic, and mixing free energies.

Adhesion Receptors Mediate Efficient Non-viral Gene Delivery

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.

Optimizing Aerosol Gene Delivery and Expression in the Ovine Lung

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

Coordinate Suppression of ERBB2 and ERBB3 by Enforced Expression of Micro-RNA miR-125a or miR-125b

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

Hydrodynamic properties of DNA and DNA–lipid complex in an elongational flow field

The aim of this study was to determine the difference between hydrodynamic properties of DNA–cetyltrimethylammonium (CTA) complex and those of DNA, which may be related to the difference in fibre-forming ability of DNA–CTA from that of DNA. Responses of DNA and DNA–CTA complex to an elongational flow field were investigated. In both solution systems, results suggesting a coil–stretch transition were obtained. From a critical strain rate value, the radius of gyration of DNA–CTA molecules in ethanol–glycerol solution was revealed to be 0.3–0.5 times of that of DNA in aqueous NaCl solution. Shear viscosity of DNA–CTA solution was much smaller than that of DNA solution, also suggesting a smaller size of DNA–CTA in ethanol–glycerol solution than that of DNA in aqueous NaCl solution. The plateau birefringence value of the DNA–CTA system, a parameter that indicates the local molecular conformation and the molecular arrangement, was only about 1/10 of that of the DNA system. There is an empirically determined molecular model of DNA–CTA complex in which a DNA molecule is sheathed by a cylindrical crust made of CTA chains. This structure reduces the DNA molecular density in a pure elongational flow field region but cannot explain the observed reduction of birefringence intensity. The small plateau birefringence value of DNA–CTA compared with that of DNA was attributed to the reduced molecular polarizability by the particular conformation of DNA molecules and CTA chains in the DNA–CTA system such as that expected by the conformational models.

Synthesis and in Vitro Evaluation of Glutamide-Containing Cationic Lipids for Gene Delivery

A novel series of cationic amphiphiles based on dialkyl glutamides with cationic pyridinium head group were synthesized as potential gene delivery agents. Four cationic lipids with glutamide as linker and varying chain lengths were tested for their transfection efficiency in three cell lines. The DNA-lipid complexes were characterized for their ability to bind to DNA, protection from nuclease digestion, size, zeta-potential, and toxicity. All four lipids demonstrated efficient transfection in MCF-7, COS, and HeLa cells, and the reporter gene expression was much higher with DOPE as the helper lipid in the formulation when compared to cholesterol. Among these 14-carbon lipids, lipid 2 has shown the highest transfection efficiency, complete protection of DNA from nuclease digestion, and low toxicity. Interestingly, lipid 2 has also shown remarkable enhancement in transfection in the presence of serum.

Electrostatic interactions across a charged lipid bilayer

We present theoretical work in which the degree of electrostatic coupling across a charged lipid bilayer in aqueous solution is analyzed on the basis of nonlinear Poisson-Boltzmann theory. In particular, we consider the electrostatic interaction of a single, large macroion with the two apposed leaflets of an oppositely charged lipid bilayer where the macroion is allowed to optimize its distance to the membrane. Three regimes are identified: a weak and a high macroion charge regime, separated by a regime of close macroion-membrane contact for intermediate charge densities. The corresponding free energies are used to estimate the degree of electrostatic coupling in a lamellar cationic lipid-DNA complex. That is, we calculate to what extent the one-dimensional DNA arrays in a sandwich-like lipoplex interact across the cationic membranes. We find that, in spite of the low dielectric constant inside a lipid membranes, there can be a significant electrostatic contribution to the experimentally observed cross-bilayer orientational ordering of the DNA arrays. Our approximate analytical model is complemented and supported by numerical calculations of the electrostatic potentials and free energies of the lamellar lipoplex geometry. To this end, we solve the nonlinear Poisson-Boltzmann equation within a unit cell of the lamellar lipoplex using a new lattice Boltzmann method.

Is the formation of cationic lipid-DNA complexes a thermodynamically driven phenomenon? Structure and phase behavior of DC-Chol/DNA complexes say not

The currently accepted mechanism of formation of cationic lipid-DNA complexes (lipoplexes) relies on the basic assumption that equilibrium structure of lipoplexes is regulated by thermodynamics. The main consequence is that neutral lipoplexes are one phase whereas positively (or negatively) charged ones coexist with excess lipid (or excess DNA). The authors report a small angle x-ray diffraction study on the structure of lipoplexes made of the cationic lipid 3β-[N-(N,N-dimethylaminoethane)-carbamoyl]cholesterol and calf thymus Na-DNA. Here the authors show that positively charged lipoplexes can coexist with unbound DNA and they claim that steric size effects are definitely important to determine the equilibrium structure of lipoplexes.

DNA Condensation and Interaction with Zwitterionic Phospholipids Mediated by Divalent Cations

Artificial viruses are considered to be a promising tool in gene therapy. To find lipid-DNA complexes with high transfection efficiency but without toxicity is a fundamental aim. Although cationic lipids are frequently toxic for cells, neutral lipids are completely nontoxic. Zwitterionic lipids do not interact with DNA directly; however, the interaction can be mediated by divalent cations. Langmuir monolayers represent a well-defined model system to study the DNA-lipid complexes at the air/water interface (quasi-2D systems). In this work, isotherms, infrared reflection absorption spectroscopy (IRRAS), X-ray reflectivity (XR), grazing incidence X-ray diffraction (GIXD), and Brewster angle microscopy (BAM) measurements are used to study the interaction of calf thymus DNA with DMPE (1,2-dimyristoyl-phosphatidylethanolamine) monolayers mediated by Ca2+ or Mg2+ ions. DNA adsorption is observed only in the presence of divalent cations. At low lateral pressure, the DNA partially penetrates into the lipid monolayer but is squeezed out at high pressure. The adsorption layer has a thickness of 18-19 A. Additionally, GIXD provides information about a one-dimensional ordering of adsorbed DNA. The periodic distance between DNA strands depends on the type of the divalent cation.

Characterization of the Residues in Helix 8 of the Human β1-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins

Several key amino acids within amphipathic helix 8 of the human beta1-adrenergic receptor (beta1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type beta1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type beta1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.

Uptake Pathways and Subsequent Intracellular Trafficking in Nonviral Gene Delivery

The successful delivery of therapeutic genes to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. Nonviral gene delivery is currently a subject of increasing attention because of its relative safety and simplicity of use; however, its use is still far from being ideal because of its comparatively low efficiency. Most of the currently available nonviral gene vectors rely on two main components, cationic lipids and cationic polymers, and a variety of functional devices can be added to further optimize the systems. The design of these functional devices depends mainly on our understanding of the mechanisms involved in the cellular uptake and intracellular disposition of the therapeutic genes as well as their carriers. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fate is usually linked to the entry mechanism. Therefore, the successful design of a nonviral gene delivery system requires a deep understanding of gene/carrier interactions as well as the mechanisms involved in the interaction of the systems with the target cells. In this article, we review the different uptake pathways that are involved in nonviral gene delivery from a gene delivery point of view. In addition, available knowledge concerning cellular entry and the intracellular trafficking of cationic lipid-DNA complexes (lipoplexes) and cationic polymer-DNA complexes (polyplexes) is summarized.

希土類錯体をドープしたＤＮＡ‐脂質複合体の光学特性

A DNA-lipid complex, where cationic surfactants are bonded to DNA, was synthesized and its optical property was examined by doping rare-earth chelates. The DNA lipid complex films to which several kinds of rare-earth chelates were doped were prepared, and their fluorescence quantum yields were examined. A 2 to 10-fold increase in quantum yield was observed as compared with conventional PMMA similarly doped. The improvement of the fluorescence quantum yield indicates that the use of DNA is promising in the development of new optical functional materials.

Oxygen Permselective Membranes from DNA-Lipid Complexes

Oxygen Permselective Membranes from DNA-Lipid Complexes

537. Charge-Reversal Lipids for Gene Transfection

One of the critical steps limiting the efficiency of non-viral gene delivery is the intracellular release of DNA from the vector complex. The complex should be stable enough to prevent DNA degradation but also dissociate from the DNA once inside the cell for subsequent transcription. To facilitate the escape of DNA from the DNA-lipid complex in the cell, and thus improve transfection efficiency, we have developed esterase sensitive cationic lipids. These lipids form a strong complex with DNA. Once in the endosome, esterases hydrolyze the terminal ester linkage of the lipid affecting a change in overall lipid charge from cationic to anionic. This charge-reversal transition releases the DNA from the lipids. These charge-reversal lipids (Fig. 1) were synthesized and characterized. The complexation and dissociation of the lipid to the DNA was monitored using an ethidium bromide displacement assay. The supramolecular lipid/DNA complexes were also characterized by dynamic light scattering, TEM and X-ray diffraction. Successful gene transfection was observed with this new class of lipids with CHO, K562, and HEK293 cells.

170. Transfected Cell Arrays for Assessment of Estrogen Receptor Activation in Breast Cancer Cells

Transfected cell arrays represent a high-throughput approach to correlate gene expression with functional cell responses, based on gene delivery from a substrate that supports cell adhesion. These arrays provide the ability to express, in parallel, thousands of exogenous genes in live cells, giving real-time information on cellular physiology and gene function. While there have been advances in transfected cell arrays, improvements are needed to this technology, including increasing the cells types that can be efficiently transfected and developing better quantification and normalization methods. We have created an array using soft lithography techniques to pattern DNA-lipid complex deposition. Specifically, a mold was fabricated by curing polydimethylsiloxane (PDMS) into thin, flat disks. Rods of precise diameters were then used to punch holes into the mold, with diameters ranging from 1 mm to 3 mm. The PDMS mold was oxidized and then reversibly sealed to polystyrene slides. The holes in the mold, termed microwells, served as reservoirs for complex deposition onto the polystyrene slides. After deposition, the PDMS mold was peeled away from the polystyrene slide, which was then rinsed thoroughly. DNA complexes were immobilized on the slide in distinct regions, replicating the pattern of microwells in the PDMS mold. Transfection of cells seeded onto these slides was also confined to the patterns.