Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA
© McLaggan et al; licensee BioMed Central Ltd. 2006
Received: 22 April 2005
Accepted: 16 January 2006
Published: 16 January 2006
Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N4,N9-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow.
Poly-APS and LipoGen were both found to be highly efficient DNA condensing agents. Fura-2 calcium imaging was used to measure calcium transients indicative of cell membrane pore forming activity. Calcium transients were evoked by poly-APS but not LipoGen and lipofectamine. The increases in intracellular calcium produced by poly-APS showed temperature sensitivity with greater responses being observed at 12°C compared to 21°C. Similarly, delivery of lucifer yellow into cells with poly-APS was enhanced at lower temperatures. Transfection with cDNA encoding for the expression enhanced green fluorescent protein was also evaluated at 12°C with poly-APS, lipofectamine and LipoGen. Intracellular delivery of siRNA was achieved with knockdown in beta-actin expression when lipofectamine and LipoGen were used as transfection reagents. However, intracellular delivery of siRNA was not achieved with poly-APS.
Poly-APS mediated pore formation is critical to its activity as a transfection reagent, but lipofection systems utilise distinct mechanisms to enable delivery of DNA and siRNA into cells.
The technologies investigated in this study involved the use of polymeric alkylpyridinium sponge toxins, (polyalkylpyridinium salts; poly-APS) [1–3]. Poly-APS is a marine toxin preparation extracted from the sponge Reniera sarai, which can form reversible pores or lesions in cell membranes . This toxin preparation primarily contains a cocktail of two polymeric 1,3-octylpyridinium salts of 5.5 and 19 kDa sizes . At a high concentration (50 μg/ml), polyalkylpyridinium salts can cause irreversible depolarisation in membrane potential and a collapse in input resistance. However, at lower concentrations (0.5 μg/ml) poly-APS show reversible actions on the electrophysiological properties and Ca2+ permeability in HEK 293 cells, dorsal root ganglion neurons and F-11 cells. The unique pore formation properties in addition to their poly-positive charged characteristics, could be useful in non-viral gene therapy (NVGT) applications. In a previous study  some us have shown that sponge toxins can be used to deliver cDNA into HEK293 cells and that this results in the expression of the protein encoded by these genes. Furthermore, the co-transfection of cDNA is stable and colonies of cells can be produced that contain poly-APS delivered cDNA and the proteins they encode for . However, there has to date been no reported study on the interaction of this polycationic toxin with DNA and further information is required on the mechanism of transfection and the versatility of macromolecule (DNA or siRNA) delivery. This is particularly important given that our data suggest membrane poration is required for poly-APS-mediated transfection because low molecular weight pyridinium surfactant monomers and dimers can also be efficient non-toxic agents for gene delivery . However, in contrast, pyridinium surfactant monomers are not efficient pore formers and appear to package DNA for delivery by fusion and/or endocytosis.
DNA condensation study of poly-APS and LipoGen
The DNA condensation experiments were run with a plasmid encoding for enhanced green fluorescent protein (pEGFP) or luciferase (pGL3), and with calf thymus DNA (ctDNA). In DNA condensation experiments, ethidium bromide (EthBr), a DNA intercalating cationic dye was used as a fluorescent probe. This intercalation increases the fluorescence yield of EthBr (excitation 546 nm, emission 595 nm). Many compounds that bind to DNA, including DNA condensing agents used in NVGT, can displace EthBr from EthBr-DNA complexes. EthBr has been widely used to assess the DNA-polyamine complex formation efficiently [9, 10]. In 2000, a modified reproducible EthBr displacement assay was reported by Geall and Blagbrough . The excitation wavelength was optimised at 260 nm  where EthBr is indirectly excited by energy transfer from the DNA. The proposed protocol also allows the experiment to be run without pre-complexing DNA and its binding molecule, a "displacement assay" . The DNA condensed particle formation has been measured at UV absorbance of 320 nm . The double-helical DNA was bound by polyamines and formed nanoparticles, which scatter the light resulting in the UV absorbance increase above 300 nm. Light scattering (LS) is measured rather than UV absorption (no chromophore for this wavelength). A little precipitation of the DNA may be visible, but it does not increase the absorption above 300 nm. The DNA concentration used in this assay was a 10-fold excess compared to the EthBr assay given the low sensitivity of this experimental system and lack of fluorescence indicator.
Poration of cell membranes by poly-APS
Intracellular delivery of lucifer yellow
pEGFP delivery by poly-APS and lipopolyamines
Intracellular delivery and function of siRNA
Given the abilities of poly-APS, lipofectamine and LipoGen to act as transfection reagents for cDNA we were interested to determine whether they would deliver siRNA into HEK 293 cells. For these experiments initial incubation protocols were carried out at 37°C because at this temperature transfection with cDNA has been achieved with all three reagents used. The intracellular delivery of siRNA was detected firstly by visualisation of intracellular siRNA labelled with fluorescein and secondly by evaluating knockdown of specific protein expression. Experiments using lipofectamine showed that in the presence of serum substantial knockdown in protein expression was observed after 48 h. In contrast when incubations were carried out in serum-free conditions substantial knockdown could be identified after 24 h but knockdown did not increase further after 48 h. Therefore in all other experiments serum-free incubation conditions were used and analysis of protein knockdown was carried out after 24 h.
Poly-APS is a highly efficient DNA condensing agent with an additional property of being able to reversibly form pores in membranes, which are highly desirable characteristics for any non-viral vectors. In addition to cell entry facilitation, this membrane perturbation could also help in endosomal escape process, which is one of key barriers in gene delivery. The structure modification of poly-APS i.e. lipid conjugation, alkyl chain length control and degree of polymerisation, may further improve its DNA condensing, pore formation, transfection efficiency, and toxicity profiles.
Other pyridinium compounds have proved useful transfection reagents, these include the SAINT (synthetic amphiphile INTeraction) series , which have high transfection efficiency when used in a liposomal formulation with 1,2-dioleoyl phosphatidylethanolamine (DOPE). The SAINT series are conjugations of two fatty acid chains (tail group) with pyridinium head groups. From structure-activity relationship studies, it was found that unsaturated fatty chains enhanced the transfection efficiency. Head group modification by introducing another pyridinium group linked with an alkyl spacer, also significantly improved the transfection efficiency. Interestingly, C4 spacer (in SAINT-3) linking two pyridinium heads shows the highest efficiency in DNA delivery, compared to C3 and C5 spacers . Lipophilicity and alkyl chain length modifications of poly-APS, leading to optimal positive charge regiochemical distribution, (distance between pyridinium head-groups in the poly-APS molecules) may in the future improve pore formation and transfection properties of modified poly-APS.
Previously, we have shown that a sponge toxin preparation, poly-APS, can produce pores in cell membranes and perform as a transfection reagent. This is unusual given that poly-APS is a natural product preparation and it raises the possibility that in nature as well as acting in chemical defence, polymeric alkypyridinium compounds may also provide a novel mechanism of natural horizontal genetic material transfer between marine microorganisms. The mechanism by which poly-APS delivers macromolecules into cells and achieves transfection is unknown. However, reversible pore formation by poly-APS and movement of macromolecules down concentration or electrochemical gradients into cells through these structures may provide a novel mechanism for the intracellular delivery of normally impermeant molecules. An alternative mechanism might involve the binding of poly-APS to cDNA, this complex binding to cell membranes and then internalisation by endocytosis. The processes of endocytosis are temperature dependent as temperature affects both rate of ligand binding and mobility of ligand-receptor complexes in membranes. Endocytotic processes are usually blocked at temperatures below 12°C. For example lucifer yellow uptake into platelets by fluid phase endocytosis occurs above 15°C . Although platelets clearly load with lucifer yellow at 37°C no such uptake was seen at this temperature in HEK 293 cells. However, dramatic stable intracellular loading of lucifer yellow into HEK 293 cells was facilitated by poly-APS during low temperature incubations. This event was not supported by lipofectamine or LipoGen and must therefore be due to some processes other than fluid phase endocytosis. Experiments were conducted to determine whether poly-APS would permeabilise cells, as reflected by calcium influx, support transfection of cDNA encoding for the expression of enhanced green fluorescent protein and delivery of lucifer yellow into cells at 7–12°C. At low temperatures, poly-APS functions well, both as a pore former and as a transfection reagent for delivery of cDNA. This indicates that endocytosis is unlikely to be the mechanism by which poly-APS delivers cDNA and lucifer yellow into cells. Additionally, the protocol used for cDNA delivery also suggests that endocytosis is not involved because there is a requirement to incubate cells in poly-APS prior to exposure with cDNA. Furthermore, the temperature data adds to the evidence (pre-mixing poly-APS with cDNA actually prevents transfection), that pore formation by poly-APS is critical for macromolecule delivery. Although lipofectamine and LipoGen could be used as a transfection reagent at 12°C they were much less efficient at low temperatures compared to high temperatures. A further difference was the failure of lipofectamine and LipoGen to cause an increase in intracellular Ca2+, therefore these reagents show very different biological activity compared to poly-APS.
The polymeric structure of poly-APS appears critical to at least some of its biological activities. We previously found that two monomeric compounds, cetylpyridinium chloride and cetyltrimethylammonium bromide, did not reversibly permeate cell membranes and did not support transfection . A recent investigation on poly-APS and synthetic linear alkylpyridinium monomers, dimers and tetramers showed that biological activities such as antibacterial, hemolytic activities, anti-acetylcholinesterase activities and inhibition of protein phosphatase 2A are greatly and differentially influenced by increasing numbers of positive charges and pyridinium rings. The smaller synthetic compounds showed good anti-enzymatic and antibacterial activity. In contrast poly-APS showed much greater hemolytic activity compared to the smaller synthetic compounds .
Evidence suggests that a reduction in temperature enhances pore formation by poly-APS both at the lowest concentration of poly-APS tested (0.1 μg/ml) and in cells that were less sensitive and failed to respond to poly-APS at room temperature. It is likely that this effect is due to both temperature-dependent alterations in physicochemical properties of the cell membrane and conformational changes in poly-APS resulting in promotion of poly-APS insertion into the membrane and pore formation. However, poly-APS did not deliver siRNA or knockdown protein, whereas both lipofectamine and LipoGen delivered siRNA and produced specific knockdown of functional β-actin similarly. It is not clear why siRNA was not delivered into HEK 293 cells.
In conclusion, poly-APS was found to be an efficient pore former and at low temperatures (7–12°C) could deliver into cells both cDNA and lucifer yellow but not siRNA. In the future it would be interesting to evaluate the binding of siRNA to poly-APS and its influences on pore formation. It is noteworthy that pyridinium salts are efficient quenching agents reflecting binding properties. Although quenching might have accounted for the negative result obtained when we attempted to deliver fluorescein-labeled siRNA with poly-APS, it does not explain the negative results with siRNA targeted against β-actin. In contrast to poly-APS, LipoGen and lipofectamine did not form Ca2+ permeant pores, but did deliver siRNA into cells and are likely to function in a similar manner. Further studies of the temperature sensitivity to poly-APS might provide approaches, using low temperature and low poly-APS concentrations, to improve efficiency and selectively delivery of macromolecules into cells. In the context of the negative temperature coefficient for poly-APS, it is interesting to note that newly discovered 1,3-dialkylpyridinium and related compounds have been extracted from Arctic species of sponges [19–21]. These findings show that alkylpyridinium compounds are not confined to temperate and tropical Haplosclerida but may contribute to host defence in cold environments.
Poly-APS (C13H20N+ monomer M.W. 190.16,  with one positive charge per monomer unit) were purified from the Adriatic marine sponge Reniera sarai as previously described , and dissolved in MilliQ water (stock solution 1 μg/μl) [1–3]. Poly-APS is readily solubilized in distilled water at room temperature and can be kept for months at 4°C without loosing biological activities. We have never observed any precipitation at 4°C when poly-APS was kept at concentrations up to 1.5 mg/ml. However, the solubility is highly dependant on the polarity of the solvent and poly-APS is only poorly soluble in methanol, and even less soluble in ethanol. There is only very limited data on phase behaviour of poly-APS in water. Experiments with dynamic and static light scattering  have shown that poly-APS exists as a mixture of monomolecular polymers, creating large supramolecular spherical aggregates with a hydrodynamic radius of 23 nm.
N4,N9-dioleoylspermine (LipoGen) [5, 7] was synthesized by the procedure described elsewhere . LipoGen was dissolved in ethyl alcohol (stock solution 2 μg/μl). Buffers and NaCl solution were also made up in MilliQ water and buffers were adjusted to 7.4 with aq. NaOH solution.
Lipofectamine was purchased from Invitrogen, U.K. pEGFP (4.7 kbp) and pGL3 (5.3 kbp) DNA were prepared according to the Maxiprep plasmid amplification and purification protocol and stored in a freezer before use. Calf thymus DNA was purchased from Sigma-Aldrich U.K. Foetal calf serum (FCS), 10 × Minimum essential media (MEM), Penicillin G, Streptomycin, L-glutamine, 7.5% NaHCO3, trypsin, PBS, and serum-free MEM (Opti-MEM) were obtained from Gibco-Invitrogen.10% Media used during transfection complexes addition was either NaCl-based saline or serum-free MEM. Other chemicals used were supplied from Sigma-Aldrich U.K.
The displacement assay fluorescence measurements were run with Perkin-Elmer Fluorescence Spectrophotometer Model LS50B (λ ex = 260 nm, λ em = 600 nm, 1 cm pathlength, 3 ml glass cuvette, slit width 5 nm) with FLWinLab version 2.00 for data processing. DNA concentration and purity were carried out with a Helios UV spectrophotometer at 260 nm (for DNA concentration) and 280 nm (for protein concentration) prior to the experiments . DNA (6 μg) was obtained from the stock solution and diluted to 3 ml with low-NaCl HEPES buffer (2 mM HEPES, 20 mM NaCl, 10 μM EDTA, pH 7.4) in a glass cuvette stirred with a micro-flea. Immediately prior to analysis, EthBr solution (3 μl, 0.5 μg/μl) was added to the DNA solution, stirred for 1 min to equilibrate the binding process. Aliquots of poly-APS or LipoGen were added to the stirring DNA solution at the desired ammonium/ phosphate (N/P) ratio and the fluorescence measured after 1 min equilibration [20, 21]. Optimal N/P ratios for DNA condensation for poly-APS and LipoGen were at 3.50–4.00 (4.1–4.7 μg/ml) and 1.50–2.50 (3.3–5.5 μg /ml). The emission intensity was reported as the percentage of maximum fluorescence (100 %) when DNA was fully intercalated by EthBr without the DNA binding agents and corrected for the background fluorescence of total EthBr in buffer solution.
For the light scattering assay, DNA (60 μg) was taken from the DNA stock solution and diluted to 3 ml with HEPES buffer (2 mM HEPES, 20 mM NaCl, 10 μM EDTA, pH 7.4) in a glass cuvette stirred with a micro-flea. Aliquots of poly-APS or LipoGen were added to the stirring DNA solution at the desired N/P ratio and the UV absorbance at 320 nm was then measured after I min equilibration .
HEK 293 cell cultures
HEK 293 cells (human embryonic kidney cell line) were maintained in culture in Dulbecco's modified Eagle's minimum essential medium, supplemented with 10 % foetal calf serum, 2 mM L-glutamine, 50 μU/ml penicillin, 50 μg/ml streptomycin and 1 % non-essential amino acids. In preparation for experiments and 24 h prior to experimentation, cells were seeded at a density of 0.5 million cells per 35 mm dish.
HtTA HeLa cell cultures
HtTA-1 HeLa cells, at 1 × 106 cells, were grown in 150 ml flask with 25 ml of 10% FCS EMEM (minimum essential media with penicillin, streptomycin, glutamine, sodium bicarbonate, and 10% foetal calf serum) [22–24]. Each 500 ml of media contained 50 ml EMEM (10×), 13.5 ml NaHCO3 (7.5%), 5 ml L-glutamine (200 mM), 25000 IU penicillin G, 25000 μg streptomycin and 10% foetal calf serum. The culture was incubated at 37°C, 5% CO2. The cells were passaged every 3 days. Trypsinized cells were added to the next passage at 1 × 106 cells/flask.
Ca2+ imaging experiments were conducted at room temperature (approximately 21°C) or 12°C. HEK 293 cells were bathed in a NaCl-based extracellular solution containing in mM: NaCl, 130; KCl, 3.0; CaCl2, 2.0; MgCl2, 0.6; NaHCO3 1.0, HEPES 10.0, glucose 5.0. The pH and osmolarity of extracellular solutions were adjusted to 7.4 and 310–320 mOsmol/l with NaOH and sucrose respectively.
Cultured HEK 293 cells were incubated for 1 h in NaCl-based extracellular solution containing 10 μM fura-2AM (Sigma, 1 mM stock in dimethylformamide) and the effects of poly-APS, lipofectamine and LipoGen on intracellular Ca2+ were evaluated using fluorescence ratiometric imaging as previously described [1–3]. For some experiments, the temperature was changed by perfusing cells with saline at 21°C and 12°C. The temperature was monitored using a thermometer probe (Jenway 2000 series) placed in the bath containing the cells. All data are given as mean ± standard error of the mean and statistical significance was determined using the Student's two-tailed unpaired t test.
Macromolecule and probe delivery protocols
cDNA delivery into HEK293 cells
HEK 293 cells were washed with NaCl-based saline or serum-free medium, then exposed to 0.5 μg/ml poly-APS for 5 min and then poly-APS and 2.5 μg/ml cDNA encoding for the expression of enhanced green fluorescent protein (pEGFP; Clontech) for 3 h at 12°C.
Additionally, experiments were conducted using lipofectamine as the transfection reagent. Lipofectamine (4 μg) was mixed with 100 μl of serum-free medium and incubated at room temperature for 5 min prior to adding to 100 μl of serum-free medium containing cDNA (1 μg/ml; N/P ratio 4). After mixing this solution containing transfection reagent and cDNA was incubated for 20 min at room temperature and then added to the cells. The cells were incubated for 3 h at 12°C. After incubation with the transfection reagent and cDNA, serum (to give 10 %) was added. For cells incubated with NaCl-based saline, serum-containing medium was added to replace the saline. Cells were maintained in culture for 24 h before green fluorescent protein was examined using either fluorescence microscopy or confocal imaging.
cDNA delivery into HtTA cells
DNA complex solution for each well was prepared from solution A and solution B. For solution A, pEGFP (2 μg) was diluted to 100 μl solution with Opti-MEM medium (serum-free media). Solution B was prepared from LipoGen (5.5 and 11.0 μg for N/P ratios of 2.5 and 5.0 respectively), or lipofectamine (2.53 μl for an N/P ratio of 3.0) was diluted to 100 μl with Opti-MEM medium. Each solution was left at 20°C for 30 min. Solutions A and B were then mixed together and vortexed for a brief period, incubated at 20°C for 20 min for DNA complexing. In 6-well or 12-well tissue culture plates, HtTA 2.5 × 104 cells/ ml were seeded in 4 ml and 2 ml of 10% FCS EMEM respectively. The cells were incubated at 37°C with 5% CO2 in an incubator for 24 h, until the cells were 30 – 50% confluent. Prior to the addition transfection complexes, media was removed and replaced with either NaCl-based saline or Opti-MEM. NaCl-based extracellular solution used in HtTA cell experiments was the same as used in HEK 293 cell experiments. For 12°C experiments, cells were placed in a temperature-controlled refrigerator for 10 min before the transfection. Solutions of DNA complexes (200 μl/well) were added, and then cells were incubated in either 37°C with 5% CO2 or at 12°C. The 48 h post-transfection samples was analysed by FACS for enhanced green fluorescent protein level.
Lucifer yellow delivery
HEK 293 cells were washed with NaCl-based saline, then exposed to 0.5 μg/ml poly-APS for 5 min and then poly-APS and 1 mM lucifer yellow (Sigma) for 1, 2 and 3 h. Pre-incubations in poly-APS and incubations in lucifer yellow were carried at 7°C, 21°C and 37°C. Cells were washed three times before intracellular loading with lucifer yellow was evaluated by fluorescence microscopy immediately after incubation and again after being placed in culture medium and cultured for a further 24 h. Additionally, the abilities of lipofectamine (4 μg/ml) and LipoGen (4 μg/ml) to load HEK 293 cells with lucifer yellow were determined over 3 h incubation periods at 7°C and 21°C.
HEK 293 cells were washed with serum-free medium, then exposed to 1.0 μg/ml poly-APS for 5 min and then poly-APS and 200 nM siRNA conjugated with fluorescein (QIAGEN) for 4 to 6 h at 37°C. An additional set of experiments were carried out with 1.0 μg/ml poly-APS and 200 nM siRNA conjugated with fluorescein (QIAGEN) for 5 h at 12°C. The fluorescence within the cells was assessed immediately after incubation using confocal imaging. Additionally, experiments were conducted using lipofectamine and LipoGen as transfection reagents. Lipofectamine (4 μg) or LipoGen (4 μg) were mixed with 100 μl of serum-free medium and incubated at room temperature for 5 min prior to adding to 100 μl of serum-free medium containing 50 nM siRNA conjugated with fluorescein. After mixing, this solution containing transfection reagent and siRNA was incubated for 20 min at room temperature and then added to the cells bathed in 1 ml serum-free medium (final volume 1.2 ml). The cells were incubated for 4–6 h at 37°C before imaging siRNA intracellular delivery.
Knockdown of β-actin protein with β-actin specific siRNA (Ambion) was assessed using Western blot analysis. HEK 293 cells were treated and incubated with transfection reagents (poly-APS, lipofectamine or LipoGen) as above with 100 nM siRNA for lipofectamine and LipoGen or with 333 μM siRNA for poly-APS (ratio 1 μg: 30 pmol siRNA for lipofectamine and LipoGen and 1 μg: 667 pmol siRNA for poly-APS) for 3 or 6 h at 37°C. The N/P values for the siRNA β-actin experiments were approximately 1.3 to 8.8 and 1.6 to 10.8 (2.4 to 0.35 μg/ml siRNA to 4 μg/ml lipofectamine or LipoGen respectively). Serum was then added (to give a final concentration of 10 %) and then the cells were maintained in culture for 24 or 48 h. siRNA negative controls were run with non-homologous siRNA. Cells were placed on ice and processed for Western blot protein analysis (Santa Cruz Biotechnology Inc.). Loading controls were conducted by stripping the blot and re-probing using antibodies for NFKB (Cell Signalling).
plasmid encoding for enhanced green fluorescent protein
non-viral gene therapy
Synthetic Amphiphile INTeraction
small interfering RNA
The authors would like to thank Professor Andy Porter, Dr Frank Gunn-Moore and Dr Juliette Snow for useful discussion. We thank Dr Bettina Platt, Dr Brian Reid, Mr Carlos Lafourcade, Mr David Koss, Ms Kathleen Hindley and Professor Graeme Nixon for help with the imaging. We also thank Mr Zhiang Zhou for help with the techniques for molecular biology and Mrs Irene Hunter and Ms Jin Pu for a preparation of HEK 293 cells. The authors are grateful to Scottish Enterprise for support under the Knowledge Transfer grants scheme. We thank Universities UK for an ORS award to Noppadon Adjimatera.
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