Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.     

Synthesis of lipid-oligonucleotide conjugates for RNA interference studies

The synthesis of RNA molecules carrying lipids at their 3'-termini and 5'-termini is reported. These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. The ability of these conjugates to silence gene expression was evaluated in the inhibition of the tumor necrosis factor. All the lipid-siRNA derivatives were compatible with RNA interference machinery if transfected with oligofectamine. In the absence of a transfection agent, some lipid-siRNA derivatives can exert a slight reduction of gene expression.     

The phase behavior of cationic lipid-DNA complexes

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram.     

The structural organization of cationic lipid-DNA complexes

The interaction of cationic liposomes with supercoiled plasmid DNA results in a major rearrangement of each component to form compact multilamellar structures comprised of alternating layers of two-dimensional arrays of DNA sandwiched between lipid bilayers. Fluorescence resonance energy transfer was used to estimate the distance of closest approach of DNA to the lipid bilayers in these complexes. The effect of several compositional variables on this distance, including the ratio of cationic lipid to DNA, and the charge density, intrinsic curvature, and fluidity of the lipid bilayer were examined. Additionally, the effect of ionic strength was studied. For complexes prepared at or above a 3:1 charge ratio (+/-), the observed distance of closest approach was found to be in agreement with the intercalation of DNA between lipid bilayers. As the charge ratio was decreased, a monotonic increase in the distance was observed with a maximum observed at 0.5:1. Correlations between differences in the proximity of DNA to the lipid bilayer and the hydrodynamic size of the complexes were also found. A model based on these observations and previous reports suggests the formation of discrete populations of complexes below a charge ratio of 0.5:1 and above 3:1. The structure of the negatively charged complexes is consistent with DNA extending from the surface of the particles, whereas those possessing excess positive charge were multilamellar aggregates with the DNA effectively condensed between lipid bilayers. Complexes between these two states consist of weighted fractions of these two species.     

Carbohydrate functionalization using cationic iron carbonyl complexes

Cationic iron carbonyl cyclohexadiene complexes were employed in the derivatization of the 3-OH position of unprotected and protected methyl beta-D-galactopyranosides using two different approaches, giving access to galactopyranosides with an aromatic or cyclohexadienoic functionality in this position.     

Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.     

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, sigma(M), for a wide range of values of the parameter (c) representing the fraction of cationic lipids. For weakly charged complexes (low values of (c)), we find that d(DNA) has a linear dependence on (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 sigma(M) halfway between adjacent DNA molecules. For highly-charged complexes (large (c)), we find moderate charge density fluctuations and observe deviations from linear dependence of d(DNA) on (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 (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.     

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.     

Multilamellar structures of DNA complexes with cationic liposomes.

Studies of DNA complexes with cationic liposomes are prompted by the search for nonviral DNA carriers for gene therapy. Recent experiments have identified a stable multilamellar phase in which ordered smectic layers of DNA alternate with cationic bilayers. In this paper we identify the forces governing DNA adsorption on cationic lamellae, including a membrane-induced attraction between the adsorbed DNA. Calculating the DNA interhelical spacing as a function of system composition, the model successfully explains recent surprising observations.     

DNA binding specificity of a series of cationic metalloporphyrin complexes

The sequence specificities of a series of cationic metalloporphyrins toward a 139 base pair restriction fragment of pBR-322 DNA have been studied by DNase I footprinting methodology. Analysis using controlled digests and quantitative autoradiography/microdensitometry revealed that the 5- and 6-coordinate complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine, MT4MPyP, where M is Mn, Fe, Co, and Zn, were found to bind to AT regions of DNA. Footprinting analysis involving the radiolabel on the opposing strand of restriction fragment showed site skewing in the direction of the 3' end of the fragment, indicating that the porphyrins bind in the minor groove of DNA. The significant increase in DNase I catalyzed hydrolysis observed in various regions of the fragment appeared to be primarily due to a decrease in available substrate DNA upon porphyrin binding with possible contributions from structural changes in DNA caused by ligand binding. The complexes NiT4MPyP and CuT4MPyP were found to bind to both AT and GC regions of the fragment, producing different degrees of inhibition in the two regions. Since the outside-binding porphyrins can neither intercalate or effectively hydrogen bond to DNA, they appear to read sequence by responding to steric and/or electrostatic potential effects located in the minor groove of DNA.     

Self-assembly of cationic palladium complexes by redistribution of pyridine ligands

The reaction of [Pd(OAc)₂(py)₂] with [Li((OEt₂)_2.5)][B(C₆F₅)₄] was conducted with intent to generate the cationic palladium complex [Pd(OAc)(py)₃][B(C₆F₅)₄], (2, py = pyridine). A single crystal structure of this material, however, reveals a 1-D polymer structure formed by the self-assembly of alternating dicationic ([Pd(py)₄]²⁺) and neutral ([Pd(OAc)₂(py)₂]) palladium units bridged by acetato linkages to give [Pd(py)₄][Pd(OAc)₂(py)₂][B(C₆F₅)₄]₂ (3). These two palladium sites are produced by disproportionation of the pyridine ligands in [Pd(OAc)(py)₃][B(C₆F₅)₄]. Proton NMR studies confirm the existence of a solvent dependent equilibrium between [Pd(py)₄]²⁺, [Pd(OAc)₂(py)₂] and [Pd(OAc)(py)₃]⁺.     

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.     

Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency

Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.     

Neutral and cationic Fe(II) β-diketiminate complexes

The three-coordinate, 12-valence electron complexes [(^MeBDK)FeR] (^MeBDK = [HC(C(Me)NAr)₂]⁻, Ar = 2,6-iPr₂C₆H₃, R = CH₂Ph, CH₂SiMe₃) are reported as well as their reactivity towards Lewis bases. With perfluoroaryl borane and -borate type activators, the monoalkyls react to give alkyl-free paramagnetic cationic iron species counterbalanced by perfluorinated arylborate anions. The paramagnetic nature of the cations permits the observation of weak and dynamic interactions with these anions via ¹⁹F NMR spectroscopy.     

Cholesterol domains in cationic lipid/DNA complexes improve transfection

The interaction between the cationic lipid DOTAP and cholesterol is examined in high cholesterol formulations by differential scanning calorimetry (DSC). Preparation of liposomes above 66 mol% cholesterol results in formulations that exhibit a calorimetric transition for anhydrous cholesterol at 38-40 °C. The enthalpy of this transition progressively increases at higher cholesterol contents, and is not detected below 66 mol% cholesterol. Furthermore, the enthalpy changes indicate that the composition of the non-domain forming portion containing DOTAP saturated with cholesterol is relatively constant above 66 mol% cholesterol. Greater transfection efficiency in the presence of 50% serum is observed at the formulations with high cholesterol contents where anhydrous cholesterol domains are detected by DSC. Although formulations possessing higher cholesterol exhibited a greater resistance to serum-induced aggregation, maintenance of small particle size does not appear to be responsible for the enhanced transfection efficiency. Additional studies quantifying albumin binding suggest that cholesterol domains in the lipid/DNA complex do not bind protein, and this may enable these moieties to enhance transfection by facilitating membrane fusion.     

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.     

Characterisation of three novel cationic lipids as liposomal complexes with DNA

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-?10-[O-(4-acetoxy)-phenylundecanoate]ethyl?- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.     

Synthesis of cationic (arene)IronCp complexes via arene exchange

Rates and regioselectivity of arene exchange reactions in cationic fused arene Fe(II)Cp complexes were investigated. Thermal exchange of pyrene, naphthalenes, and cyclooctatetraene occurs in the temperature range of 90-140 °C. The most labile complex in the series studied is [(η⁶-(1-4,4a,8a)-1,4-dimethoxynaphthalene)FeCp][PF₆] having the FeCp coordinated to the substituted ring. Pyrene and other naphthalene complexes come next, followed by the cyclooctatetraene complex. Phenanthrene, veratrol, and dihydronaphthalene do not undergo exchange at temperatures up to 130 °C. With Me- and OMe-substituted naphthalenes, exchange is reversible and favors the product having the metal coordinated to the non-substituted ring. The X-ray crystal structures of the two regioisomeric 1,4-dimethoxynaphthalene complexes were determined. Arene exchange in fused arene complexes is shown to be a useful synthetic method and provides new arene complexes cleanly and efficiently. The method is particularly attractive for arenes that contain functionalities that are not compatible with the Lewis acid-mediated routes. The starting materials are readily accessible via the TiCl₄-assisted Cp exchange in ferrocene.     

Preparation, stability and antimicrobial activity of cationic cross-linked starch-iodine complexes

Cationic cross-linked starch (CCS)-iodine complexes containing different amounts of quaternary ammonium groups (different degrees of substitution (DS)) and iodine have been obtained by iodine adsorption on CCS from aqueous iodine potassium iodide solution. Equilibrium adsorption studies showed that with an increase of DS the amount of iodine adsorbed on CCS and the affinity of iodine to CCS increased linearly. The influences of the DS of CCS and the amount of adsorbed iodine on the stability of CCS-iodine complexes in a solution of 0.02M sodium acetate and reactivity toward l-tyrosine have been investigated. At the same DS, the stability of CCS-iodine complexes decreased with an increase of the amount of adsorbed iodine. With increasing the DS, the stability of CCS-iodine complexes increased. The iodine consumption in the reaction with l-tyrosine increased significantly with an increase of the amount of adsorbed iodine. The influence of DS on iodine consumption was lower and depended on the amount of adsorbed iodine. The antibacterial activity of CCS-iodine complexes against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was determined by the broth-dilution and spread-plate methods. The obtained results have demonstrated that an appropriate selection of the CCS-iodine complex composition (the DS of CCS and the amount of adsorbed iodine) could ensure good antimicrobial properties by keeping a low concentration of free iodine in the system. The main advantage of using CCS-iodine complexes as antimicrobial agents is the biodegradability of the polymeric matrix.