Plasmid DNA-encapsulating liposomes: Effect of a spacer between the cationic head group and hydrophobic moieties of the lipids on gene expression efficiency

We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-l-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery.     

Influence of the spacer of cationic gemini amphiphiles on the hydration of lipoplexes

The impact of the length of gemini surfactant spacer on complexation and condensation of calf thymus DNA by cationic mixed phospholipid/gemini liposomes was investigated by monitoring the conformational changes of DNA by circular dichroism and the lipid hydration level by the emission characteristics of the fluorescent probe laurdan included in the lipid bilayer. The length of the spacer was shown to influence, on one hand, the hydration level and the organization of the corresponding liposomes and, on the other, the variation of lipid hydration level and the DNA conformation upon complexation. In fact, in correspondence with the longest spacer we observed more hydrated liposomes, probably organized in domains, a higher extent of dehydration promoted by the addition of DNA, and a minor extent of DNA conformational change. The physicochemical features of lipoplexes were shown to depend on the [cationic headgroup]/[DNA single base] ratio.     

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.     

Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.     

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.     

Targeted Delivery of Nucleic Acids by Folate-Containing Liposomes into KB-3-1 and HEK 293 Cells

Russian Journal of Bioorganic Chemistry - Targeted cationic liposomes containing folate lipoconjugates with spacer groups of various lengths and natures were prepared; their physicochemical...     

Spacer structure and hydrophobicity influences transfection activity of novel polycationic gemini amphiphiles

Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine^® 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery.     

Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei.

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.     

Occurrence of 9 homologous repeat units in the external spacer region of a nuclear maize rRNA gene unit.

A 14 kb maize DNA fragment carrying nuclear rRNA genes and spacer regions was isolated and characterised by restriction enzyme mapping. A complete 3020 bp long external spacer region was sequenced and revealed 9 tandemly arranged 200 bp long repeat units with high homology. The repeat units lie upstream from two prominent S1 mapping signals. The sequence of a typical repeat unit is compared to a corresponding 130 bp long wheat repeat unit. The possible functional relevance of the repeat units is discussed.     

Frequent duplication and deletion events in the 5S RNA genes and the associated spacer regions of theTriticeae

The 5 S DNA units from 15 grasses in theTriticeae were analysed at the DNA sequence level. Four units carried duplications near the 3-end of the 5 S RNA gene with 3 of the duplications centred on the same base pairs as a duplication previously reported byGerlach & Dyer. The fourth duplication was located 3 downstream from the gene, in the spacer region. Apparent deletions were very frequent when units of the different grasses were compared and it was clear that these deletions did not extend into a 75 bp spacer region upstream from the 5 S RNA gene. This 75 bp region also tended to be more conserved between the grasses as compared to the high level of sequence change in the rest of the spacer region. — Phenetic relationships were established between the grasses using the sequence data. The relationships were generally consistent with the data from other parameters and, in addition, showed that two Australian grasses were closely related to the other Northern hemisphere genera examined. The data concerning the Australian grasses is discussed in relation to the isolated nature of Australia.     

Threshold effects on the lectin-mediated aggregation of synthetic glycolipid-containing liposomes

Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a β-galactoside the liposomes are aggregated by ricin and when it is an α-mannoside they are aggregated by Con A. The lectin-mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin-mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on (1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl-containing glycolipid is independent of glycolipid concentration, and (2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane-bound receptors in order for aggregation to occur is discussed.     

Proton transport through a peptide-tethered bilayer lipid membrane by the H(+)-ATP synthase from chloroplasts measured by impedance spectroscopy.

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.     

Trypsin purification by affinity binding to small unilamellar liposomes

A novel protein purification process using affinity-ligand-modified liposomes and membrane ultrafiltration is described. The feasibility of the process was tested using trypsin as the model protein and p-aminobenzamidine (PAB) as the affinity ligand for trypsin. The affinity liposomes were prepared by covalently attaching PAB to the surface of small unilamellar liposomes via the hydrophilic spacer arm diglycolic acid. The liposomes were comprised of dimyristoyl phosphatidyl choline, cholesterol, and dimyristoyl phosphatidyl ethanolamine to which the diglycolic acid was attached. The equilibrium binding constant between trypsin and immobilized PAB was shown to be dependent on the PAB density of the liposome surface. Bound trypsin was eluted from the liposomes by the trypsin inhibitor benzamidine. Trypsin was purified from a trypsin/chymotrypsin mixture and from one of its naturally occurring sources, porcine pancreatic extract. A recovery yield from the crude mixture of 68% was obtained with a trypsin purity of 98%. The affinity-modified liposomes were stable in the complex mixture and retained their trypsin binding capacity after multiple adsorption/elution cycles over a 30-day period.     

Studies on protein-liposome coupling using novel thiol-reactive coupling lipids: influence of spacer length and polarity.

To optimize the preparation of immunoliposomes, we investigated the coupling of thiolated IgG and BSA to liposomes using a novel group of coupling lipids. All lipids consist of cholesterol as membrane anchor and a thiol-reactive maleimide headgroup, linked by a spacer that differs in length and polarity (ethylene glycol, tetraethylene glycol, PEG 400, PEG 1000, dodecyl). In addition, lipids differ in the electrophilicity of the maleimide group (p- or m-maleimidobenzoic ester). In the case of BSA, coupling efficiency strongly depended on the electrophilicity of the maleimide group as well as on the spacer polarity: The less electrophilic meta constitution seems to be an advantage over the p-maleimidobenzoic ester, resulting in higher coupling efficiency. Polar spacers (tetraethylene glycol, 46%) achieved a higher coupling efficiency than a nonpolar spacer with approximately the same length (dodecyl, 15%).When liposomes containing coupling lipids with the spacers tetraethylene glycol, PEG 400, and PEG 1000 were linked to BSA, coupling efficiencies were in a medium range and similar (41-46%) but were lower for the short ethylene glycol spacer (30%). In contrast, for IgG coupling efficiencies correlated with increasing spacer length. Best results were obtained using coupling lipids with a long polar spacer (PEG 1000) (65%), whereas a coupling lipid bearing a short spacer (ethylene glycol) resulted in a low coupling efficiency of 12%.     

Comparison of the nuclear ribosomal units of five Chlamydomonas species

The organization of the nuclear ribosomal units of five different species of Chlamydomonas has been examined by hybridization of their nuclear DNA fragments produced by several restriction endonucleases with a radioactively labelled probe consisting of the two cloned BamHI ribosomal fragments of C. reinhardii. The results indicate that a) the ribosomal units of these five species are structurally related, b) changes in the non transcribed spacer occur in C. eugametos and in C. globosa, c) the rDNA unit of C. intermedia contains either an enlarged internal transcribed spacer or a ribosomal intervening sequence, d) the rDNA units of C. reinhardii and C. callosa are indistinguishable.     

The large spacer of a nuclear ribosomal RNA gene from radish: organization and use as a probe in rapeseed breeding

Summary An Eco RI fragment spanning the spacer region separating two adjacent radish rDNA units was isolated and partially characterized. Although previous studies did not reveal obvious length heterogeneity in radish rDNA units, we observed the presence of several short repeats within this spacer, thus demonstrating that these repeats are not typical of species with variable length rDNA spacer. A short fragment containing two and one-half repeats was sequenced and used as a probe to demonstrate that this short sequence is highly specific for the genus Raphanus. We used these rDNA spacer sequences in preliminary assays for variability among 14 rapeseed cultivars and for introgression of radish rDNA in rapeseed to illustrate the usefulness of these probes.