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.     

Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts

SPARC (secreted protein, acidic and rich in cysteine), also known as osteonectin, is an extracellular Ca+2-binding glycoprotein that inhibits the incorporation of [3H]-thymidine and delays the onset of S-phase in synchronized cultures of bovine aortic endothelial (BAE) cells. This effect appears not to be dependent on the functional properties of SPARC associated with changes in cell shape or inhibition of cell spreading. In this study we investigate the conditions under which cell cycle modulation occurs in different types of cells. Human umbilical vein endothelial cells, a transformed fetal BAE cell line, and bovine capillary endothelial cells exhibited a sensitivity to SPARC and a cationic peptide from a non-Ca+2-binding region of SPARC (peptide 2.1, 0.2-0.8 mM) similar to that observed in BAE cells. In contrast, human foreskin fibroblasts and fetal bovine ligament fibroblasts exhibited an increase in the incorporation of [3H]-thymidine in the presence of 25 microM-0.2 mM peptide 2.1; inhibition was observed at concentrations in excess of 0.4 mM. This biphasic modulation could be further localized to a sequence of 10 amino acids comprising the N-terminal half of peptide 2.1. A synthetic peptide from another cationic region of SPARC (peptide 2.3) increased [3H]-thymidine incorporation by BAE cells and fibroblasts in a dose-dependent manner. In endothelial cells, a stimulation of 50% was observed at a concentration of 0.01 mM; fibroblasts required approximately 100-fold more peptide 2.3 for levels of stimulation comparable to those obtained in endothelial cells. The observation that SPARC and unique SPARC peptides can differentially influence the growth of fibroblasts and endothelial cells in a concentration-dependent manner suggests that SPARC might regulate proliferation of specific cells during wound repair and remodeling.     

Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.     

Lipitoids--novel cationic lipids for cellular delivery of plasmid DNA in vitro

To determine whether or not the erythrophore originates from xanthophores in the dorsal skin of the brown frog, Rana ornativentris, we morphologically examined the differentiation and migration of the two chromatophore types and their pigmentary organelle formation. At an early tadpole stage, three kinds of chromatophores, xanthophores, iridophores, and melanophores, appeared in the subdermis, whereas the erythrophore did so just before the foreleg protrusion stage. By the middle of metamorphosis, most chromatophores other than erythrophores had migrated to the subepidermal space. Erythrophores, which appeared late in the subdermis, proliferated actively there during metamorphosis and finished moving into the subepidermal space by the completion of metamorphosis. Carotenoid vesicles and pterinosomes within the erythrophores and xanthophores showed several significant differences in structure. In xanthophores, carotenoid vesicles were abundant throughout life, whereas those in erythrophores decreased in number with the growth of the frogs. The fibrous materials contained in the pterinosomes were initially scattered but soon formed a concentric lamellar structure. In erythrophores, the lamellar structure began to form at the periphery of the organelles but at the center in xanthophores. In addition, the pterinosomes of erythrophores were uniform in size throughout development, while those of xanthophores showed a tendency to become smaller after metamorphosis. The pterinosomes of xanthophores were significantly larger than those of erythrophores. These findings suggest that an erythrophore is not a transformed xanthophore, although they resemble each other closely in many respects.     

铕三元配合物的合成、表征及其光致发光性能

以苯甲酸、邻苯二甲酸、间苯二甲酸、水杨酸、丙烯酸、甲基丙烯酸、α—噻吩基三氟甲酰丙酮为第一配体,二安替比林甲烷,三正辛基氧化膦、2,2’-联吡啶、邻菲咯啉及邻菲咯啉N-氧化物为第二配体,合成了系列铕三元配合物。经元素分析确定了它们的组成;研究了它们的紫外吸收光谱、红外吸收光谱及荧光光谱。紫外光谱的研究表明,配合物的紫外吸收主要表现为配体的吸收,但是吸收峰的位置发生了移动;红外光谱的研究表明,配合物的红外光谱不同于自由配体的红外光谱,在400~500 cm-1出现了吸收峰,这是Eu-O的伸缩振动峰;荧光光谱的研究表明,第二配体的加入可以显著提高配合物的荧光性能。     

Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.     

Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability

PURPOSE: Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. METHODS: Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. RESULTS: In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time. from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. CONCLUSIONS: Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.     

The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes

Four cationic polymers used to deliver DNA into cultured cells: polylysine, intact polyamidoamine dendrimer, fractured polyamidoamine dendrimer and polyethylenimine, are examined for their ability to interact with DNA. Complexes between the polymers and DNA were examined using electron microscopy. Similar toroidal structures with diameters of 55 +/- 12 nm were formed from all of the cationic polymers with DNA. The DNA complexes of the fractured dendrimer and polyethylenimine were observed as single, distinct units; their apparent diameters in solution as measured by dynamic light scattering ranged from 90 to 130 nm. The DNA complexes of polylysine and intact dendrimer generally appeared as clusters in electron micrographs; their diameters in solution were larger than 1000 nm, which suggests that their toroidal complexes aggregate in solution. The cationic polymers bind to DNA in a stoichiometry that is nearly 1:1 in primary amines to DNA phosphates. The apparent binding of all cationic polymers to DNA decreases linearly with increasing ionic strength, up to 0.8 M NaCl. Thus, at the concentrations studied, these polymers interact electrostatically with DNA forming a unit structure with toroidal morphology; the extent of aggregation of the unit structures in solution depends upon the characteristics of the individual polymer.     

Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer

The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.     

The effect of vincristine-polyanion complexes in STEALTH liposomes on pharmacokinetics, toxicity and anti tumor activity

Poly(ethylene glycol) (PEG)-derivatized liposome vehicles improve antitumor effectiveness of entrapped anthracyclines and vinca alkaloids. However, the plasma clearance of entrapped vincristine is substantially faster than the lipid phase or other entrapped aqueous markers, suggesting leakage out of the liposome during transit in the blood compartment. We tested the effect of altering the drug's in vivo leakage rate on pharmacokinetics, toxicity, and antitumor activity of entrapped drug in rodent models. Suramin, heparin, and dextran sulfate were tested for their ability to produce a precipitable complex in vitro. PEG-derivatized liposomes were prepared with the complexing agent inside, and vincristine was driven inside using an ammonium gradient. The resulting preparations were found to have plasma distribution half-lives significantly longer than the formulation without a complex-forming agent. There was no increase in acute lethality, and in the case of the suramin-vincristine complex, the acute lethality was significantly reduced at the highest does level. Anti-tumor activity against the mouse mammary carcinoma MC2 was tested in a multiple-dose study. Free vincristine did not affect the tumor growth rate significantly, but at the same dose level all PEG-coated liposome formulations inhibited tumor growth markedly. The suramin containing formulation was as effective as the formulation lacking polyanion, but the heparin and dextran sulfate containing formulations were less effective. Thus, compounds which form insoluble complexes with vincristine alter in vivo plasma distribution phase pharmacokinetics without increasing acute lethality, but without a corresponding increase in anti-tumor activity.     

Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site. Implications for the role of lysine 136 in DNA binding

We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine epsilon-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.     

Innovative strategies for the oral delivery of drugs and peptides

Conventional forms of administration for nonabsorbable drugs and peptides often rely on parenteral injection, because the intestinal epithelium represents a major barrier to the oral absorption of these therapeutic agents. Recently, a number of innovative drug-delivery approaches have been developed, including entrapment within small vesicles and passage through the space between adjacent intestinal cells. This article reviews some of the most promising techniques currently available for oral delivery and their possible practical applications for the delivery of vaccines and drugs for the treatment of clinical conditions that require frequent, chronic parenteral administration.     

Modulation of the catalytic rate of Cu,Zn superoxide dismutase in single and double mutants of conserved positively and negatively charged residues

The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Glu131, and Lys134, has been determined by pulse radiolysis as a function of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutralization of Asp130, while neutralization of either of the two lysines drastically decreases the enzyme activity. The Lys120Leu-Lys134Thr and Lys134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives rise to a faster enzyme at any ionic strength value. The effects observed in the single Asp130Gln and Lys120Leu mutants differ from those reported on human or bovine enzymes [Getzoff et al. (1992) Nature (London) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indicating that some residues occupying the same position in the linear sequence of different Cu,Zn superoxide dismutases have a different functional weight. Our results also suggest that the strategy of multiple charge mutation may be a promising approach in order to increase the catalytic rate of Cu,Zn SODs independently of ionic strength.     

Selective recognition of tandemly repeated DNA sequences by homo- and heterodimers of short peptides

The non-covalent and specific protein-protein interaction is critical to the specificity and the cooperativity of the DNA-binding by protein dimers. We have designed and synthesized three sets of peptide dimers with covalent- or noncovalent artificial dimerization modules to elucidate the structural and thermodynamic aspects for the sequence-specific DNA-binding by protein dimers. We have investigated the DNA binding of covalent dimer, noncovalent homodimer and noncovalent heterodimer with specific or nonspecific DNA sequences by gel mobility shift assay. Although the amino acid sequence of DNA-binding region of these peptide dimers are the same, the selectivity and the cooperativity of DNA binding by these peptide dimers were found to be different.     

Photosensitized formation of 8-hydroxy-2'-deoxyguanosine and DNA strand breakage by a cationic meso-substituted porphyrin

Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA. In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic porphyrin. The data demonstrated a dose-dependent formation for each type of DNA damage. Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage. However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D2O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process. These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity.     

ATP-dependent assembly of a ternary complex consisting of a DNA mismatch and the yeast MSH2-MSH6 and MLH1-PMS1 protein complexes

MSH2 and MSH6 proteins exist as a stable complex, as do the MLH1 and PMS1 proteins. To study the mismatch binding properties of the MSH2-MSH6 complex and to examine its functional interaction with the MLH1-PMS1 complex, these protein complexes were purified to near homogeneity from overproducing yeast strains. As has been reported previously, the purified MSH2-MSH6 complex binds DNA substrates containing a G/T mismatch and insertion/deletion mismatches, but the binding affinity for the latter decreases as the size of the extrahelical loop increases. Addition of ATP or the nonhydrolyzable ATPgammaS reduces binding of the MSH2-MSH6 complex to the DNA substrates markedly. Here, we show that MSH2-MSH6 forms a ternary complex with MLH1-PMS1 on a mismatch containing DNA substrate. The formation of this ternary complex requires ATP, which can be substituted by ATPgammaS, suggesting that ATP binding alone is sufficient for ternary complex formation. Thus, it appears that ATP binding by the MSH2-MSH6 complex induces a conformation that is conducive for the interaction with MLH1-PMS1 complex, leading to the formation of the ternary complex.