The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Solid Supported Vesicles for Bactericide Delivery

Abstract

Cationic and anionic liposomes have been prepared by extrusion from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with cholesterol and dimethyldioctadecyltrimethylammonium bromide (DDAB) and with phosphatidylinositol (PI) respectively covering a range of composition from 0 to 19 mole % DDAB and PI. The adsorption of liposomal lipid from the liposome dispersion onto particles of silica and titanium dioxide in suspension has been studied as a function of liposome composition and concentration. The adsorption isotherms have been fitted using a Langmuir equation from which the binding constants and maximum surface coverage were obtained. The Gibbs energies of adsorption for the cationic liposomes were on average -61.0 ± 2.1 kJ mol^?1 (on silica) and -50.6 ± 2.9 kJ mol^?1 (on titanium dioxide). On average saturation adsorption is equivalent to 3 to 10 lipid monolayers on silica and 3 to 7 on titanium dioxide. Using liposomes encapsulating D-glucose it is demonstrated that there is almost no release of glucose on adsorption of the lipid, indicating that the liposomes are adsorbed intact to form a liposome monolayer on the particle surfaces. Adsorption of intact liposomes to form a close-packed liposome monolayer of solid supported vesicles (SSV) is shown to be equivalent to on average 7.0 ± 0.2 phospholipid monolayers. The SSVs are shown to have increased stability to disruption by surfactants and when carrying the oil-soluble bactericide, Triclosan?, to be capable of inhibiting the growth of oral bacteria from immobilised biofilms.     

The effect of shear on the desorption of liposomes adsorbed to bacterial biofilms

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,beta[N-(N1,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immobilized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome-biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

The delivery of benzyl penicillin to Staphylococcus aureus biofilms by use of liposomes

The delivery of benzyl penicillin [penicillin G (pen-G)] encapsulated in cationic liposomes to a pen-G-sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter approximately 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC-chol) at a molar ratio of 1.0:0.49:0.43. This composition containing 22 mole% of the cationic lipid DC-chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561-570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen-G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen-G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 x 10- 3 mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen-G. The results were supported by direct measurements of the distribution of pen-G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen-G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.     

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

The Delivery of Benzyl Penicillin to Staphylococcus aureus Biofilms by Use of Liposomes

The delivery of benzyl penicillin [penicillin G (pen‐G)] encapsulated in cationic liposomes to a pen‐G‐sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter ～ 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC‐chol) at a molar ratio of 1.0 :0.49 :0.43. This composition containing 22 mole% of the cationic lipid DC‐chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561–570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen‐G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen‐G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 × 10^{? 3} mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen‐G. The results were supported by direct measurements of the distribution of pen‐G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen‐G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Nonuniform spatial patterns of respiratory activity within biofilms during disinfection.

Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Expression of luciferase plasmid (pCMVLuc) entrapped in DPPC/Cholesterol/DDAB liposomes in HeLa cell lines

The luciferase gene expression of lipoplexes, a liposome containing luciferase plasmid (pCMVLuc), in HeLa cell lines, was investigated. Cationic liposomes were prepared by the chloroform film method with sonication. The lipoplex was formed by loading the liposome with pCMVLuc. The lipoplex with an optimal weight ratio of dimethyl dioctadecyl ammonium bromide (DDAB)/pCMVLuc protected from DNaseI was determined by an agarose gel electrophoresis. The selected lipoplexes were assayed for luciferaase activity by using a luminometer. The effect on cell proliferation was evaluated by WST-1 assay. The highest luciferase activity of 1.5 × 10⁶ RLU was observed in the cholesterol (Chol)/DDAB (2:1 molar ratio) lipoplex at the DDAB/pCMVLuc weight ratio of 10:1 at 48 hours, which was about 10, 100, and 1,000 times higher than the DDAB, L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/Chol/DDAB (1:2:1 molar ratio), and DPPC/Chol/DDAB (2:2:1 molar ratio) lipoplexes, respectively. The liposome with the smallest particle size was obtained from the cationic liposome composed of DPPC/Chol/DDAB (7:1:1 molar ratio) with the ζ potential of 7.17 ± 0.73. The optimal weight ratio of DDAB/pCMVLuc that protected pCMVLuc from DNaseI digestion was 4:1 in the DDAB formulation. The Chol/DDAB (2:1 molar ratio) lipoplex with the DDAB/pCMVLuc of 10:1 showed the highest luciferase activity of 1.5 × 10⁶ RLU and the highest cytotoxicity as well. DPPC/Chol/DDAB (1:1:1 molar ratio)-lipoplex (DDAB/pCMVLuc = 14:1), which had the amount of DPPC and cholesterol not exceeding 33 and 50% mol, respectively, gave the lower gene expression of about 4 times, but lower cytoxicity of about 14 times, than the Chol/DDAB lipoplex (2:1 molar ratio) and was considered to be the most suitable formulation. The results from this study can be applied as a model for the development of a gene-therapeutic dosage form.     

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms

Context: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail.

Objective: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms.

Materials and methods: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were ?13 and 8?mV, respectively, and both had a mean particle size of approximately 180?nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy.

Results: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms.

Discussion and conclusion: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.     

Interactions between non-phospholipid liposomes containing cetylpyridinium chloride and biofilms of Streptococcus mutans: modulation of the adhesion and of the biodistribution

Cetylpyridinium chloride (CPC) is a surfactant that binds strongly to bacteria and bacterial biofilms. In this study, fluorescence-based techniques were used to determine the penetration and adhesion of CPC when it was introduced in liposomes. In spite of a reduced adhesion as compared to pure CPC micelles, CPC-containing liposomes adhered significantly to the biofilms of Streptococcus mutans. In contrast, no binding was observed for liposomes that were composed of phosphatidylcholine-cholesterol. The influence of the charge of the liposome on its adhesion to biofilms was studied using cholesterol (Chol) and cholesterol sulfate (Schol). In spite of similar binding to the biofilms, positively charged CPC/Chol liposomes were located mainly in the core of the biofilm microcolonies, whereas the negatively charged CPC/Schol liposomes were mainly concentrated at their periphery. This effect may be attributed to the different availability of the CPC head group. In summary, this work demonstrates the high potential for tailoring drug nanovectors by modulating sterol selection in order to selectively target and bind biofilms.     

Evaluation of Fleroxacin Activity against Established Pseudomonas fluorescens Biofilms

Scanning confocal laser microscopy (SCLM) and fluorescent molecular probes were used to evaluate the effect of the fluoroquinolone fleroxacin on the architecture of established Pseudomonas fluorescens biofilms. Control P. fluorescens biofilms were heterogeneous, consisting of cell aggregates extending from the attachment surface to maximum measured depths of ~90 μm (mean biofilm depth at 72 h, 42 ± 28 μm) and penetrated by an array of channels. In contrast, fleroxacin-treated biofilms were less deep (mean biofilm depth at 72 h, 29 ± 8 μm), varied little in depth over large areas, and consisted of a homogeneous distribution of cells. Fleroxacin also caused cells to elongate, with cells located near the biofilm-liquid interface lengthening significantly more than cells located at the attachment surface. By using SCLM, acridine orange, and image analysis it was found that ~59% of cells within fleroxacin-treated biofilms emitted red fluorescence whereas >99% of cells from control biofilms emitted green fluorescence. The fleroxacin-treated cells which emitted red fluorescence were observed to be the population of cells which elongated.     

Liposomal formulations of protein proteinase inhibitors: Preparation and specific activity

Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin) activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI and BPTI delivery.     

A method to determine the incorporation capacity of camptothecin in liposomes

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.