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Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer. Despite intensive research activity, the aim of creating a vector which meets all necessary demands has still not been reached. One possibility to solve the nonviral vector associated problem of low transfection efficacy is the development of new cationic amphiphiles. Therefore, the non-glycerol-based cationic lipids 1-9 have been synthesized and tested for in vitro gene delivery experiments. The backbone structure of the lipids consists of a malonic acid diamide with two long hydrophobic chains. The degree of saturation of the hydrophobic chains and the structure of the polar cationic headgroup were varied. The preparation follows an easy process and facilitates the trouble-free insertion of different alkyl chains. By studying in vitro gene delivery an increase of transfection efficacy was observed when using at least one unsaturated alkyl chain in the hydrophobic part and lysine or bis(2-aminoethyl)aminoethylamid as hydrophilic headgroup. This leads to cationic lipids exhibiting comparable or even higher transfection efficacies compared to the commercially available LipofectAmine and SuperFect.

The feasibility of using nanoliposomes as an oral delivery system to improve the intestinal absorption of coenzyme Q(10) (CoQ(10), ubiquinone-10) was examined using Caco-2 cell monolayers as the model of human intestinal epithelium. The apparent permeability coefficient of CoQ(10) with nanoliposomes as vehicles was increased to be 4.19 +/- 0.76 x 10(-6) cm/s, which was higher than the favorable intestinal absorption value (1 x 10(-6) cm/s). The kinetic data demonstrated that nanoliposomal CoQ(10) transport occurred via passive diffusion and carrier mediation routes. Nanoliposomes might suppress the modulation activity of the peptide transporter, organic anion transporter, and P-glycoprotein through fluidizing cell membrane. The transported CoQ(10) was still in the original oxidative form as ubiquinone-10. However, 70-80% of coenzyme Q(10) accumulated by the cells was in the reduced form (ubiquinol-10, CoQ(10)H(2)), suggesting that the conversion of CoQ(10) to CoQ(10)H(2) takes place in the enterocytes during its absorption. These results indicated that the absorption behavior of CoQ(10) was modified through nanoliposomes. The bioavailability of CoQ(10) following its oral administration might be improved with nanoliposomes as the delivery system.

The high biocompatibility and versatile nature of liposomes made these particles keystone components in many hot-topic research areas. For transfection and cell labelling purposes, synthetic cationic lipids are often added, but in most studies, little attention has been paid to their cytotoxic effects. In the present work, cationic magnetoliposomes (MLs), i.e. iron oxide cores enwrapped by a phospholipid bilayer (dimyristoylphosphatidylcholine or sphingomyelin) doped with cationic lipids (1,2-distearoyl-3-trimethylammonium propane), serve as a model to examine cationic lipid toxicity. Mechanisms of cytotoxic effects were found to be either dependent or independent of actual particle internalisation according to data obtained in the absence or presence of several endocytosis inhibitors. The former seem to be caused by the generation of reactive oxygen species (ROS) leading to a Ca2+ influx at high ROS levels. The latter are due to a destabilisation of the cell plasma membrane upon transfer of the cationic lipid from the ML bilayer into the plasma membrane. However, these adverse effects can be diminished by the use of a ROS scavenger, a Ca(2+)-channel blocker or by modulating the liposome size, lipid bilayer constitution or by stabilising the membrane by anchoring it on a solid core. Careful attention must be paid in terms of assessing cell viability as the effects are highly time dependent and the data suggest the incompatibility of using the well-known MTT assay when high levels of ROS species are generated.

The pulmonary insulin delivery characteristics of liposomes were examined. Aerosolized liposomes containing insulin were administered into rat lungs and the enhancing effect on insulin delivery was evaluated by changes of plasma glucose levels. Liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhanced pulmonary insulin delivery in rats, however, liposomes with dilauroyl, dimyristoyl, distearoyl or dioleoyl phosphatidylcholine did not. Liposomes with DPPC also enhanced the in vitro permeation of FITC dextran (Mw 4400, FD-4) through the calu-3 cell monolayer by reducing the transepithelial electrical resistance and did not harm lung tissues in rats. These findings suggest that liposomes with DPPC enhance pulmonary insulin delivery by opening the epithelial cell space in the pulmonary mucosa not mucosal cell damage. Liposomes with DPPC could be useful as a pulmonary delivery system for peptide and protein drugs.

We newly prepared lipopolyplexes containing N-lauroylsarcosine (LS) as a hybrid vector for pulmonary gene delivery via the systemic route. Lipopolyplexes were composed of polyethylenimine (PEI), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA), LS, and plasmid DNA (pDNA). The particle size of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was not largely changed by the addition of LS, although the addition of LS decreased the high zeta potential. Lipopolyplexes containing LS with low zeta potential showed little aggregation with erythrocytes and low cytotoxicity. In HepG2 cells, lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D. After intravenous injection of the complexes into mice, lipopolyplexes containing LS showed high transgene efficiency, comparable to lipopolyplex 2P-2D. In particular, lipopolyplexes containing LS showed extremely high transgene efficiency in the lung. As a result of the analysis to identify optimum formulations, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index. These results suggest that lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity.

The purpose of this study was to evaluate the potential of submicron-sized liposomes (ssLips) as a novel system for delivering ocular drugs to the eye's posterior segment. Fluorescence emission of coumarin-6 formulated into ssLip was obvious in that segment in mice after eyedrop administration of the liposomal suspension. Such fluorescence was not observed after administration of either multilamellar vesicles or dimethyl sulfoxide (DMSO) solution containing the same amount of coumarin-6. The highest fluorescence of ssLip occurred 30 min after eyedrop administration, and all fluorescence disappeared after 180 min. The ssLip based on l-alpha-distearoyl phosphatidylcholine (DSPC ssLip) showed higher fluorescence emission in the retina than that based on egg phosphatidylcholine (EPC ssLip). These results confirmed that the magnitude of fluorescence in the retina was closely related to both liposome rigidity and particle size. Images of the entire eye showed that ssLip was delivered via the non-corneal pathway after administration. The liposomes tested in ocular cells showed little cytotoxicity. These results suggest that ssLip can be used to deliver drugs to the posterior segment of the eye.

The preparation of Chitosan (CHT)-coated liposomes and their applicability as a carrier for delivery of drugs to the lungs by nebulisation was investigated. Empty SUV (small unilamellar) liposomes were initially prepared (with different lipid compositions) and coated with CHT by dropwise addition of CHT solution in the liposome dispersion. CHT-coating efficiency was calculated after separation of coated/non-coated liposomes by centrifugation, and measurement of lipid in each fraction. After establishing the best conditions for CHT-coating (concentration of CHT in the solution), RIF-loaded CHT-coated liposomes, with different lipid compositions (negatively charged and non-charged) were constructed, and their encapsulation efficiency (EE) and nebulisation efficiency (NE%)/stability (NER%) were evaluated. Charged liposomes (containing phosphatidylglycerol [PG]) can be coated with CHT better compared to non-charged ones. The EE of CHT-coated liposomes (that contain PG) is slightly increased while their stability after nebulisation is significantly increased (NER%). Mucoadhesive properties of CHT-coated liposomes were substantially better (compared to non-coated ones) while the toxicity of liposomal RIF towards A549 epithelial cells was lower compared to free drug for all the types of vesicles evaluated, and especially the CHT-coated ones. Thereby, it is concluded that CHT-coated liposomes have advantages (compared to non-coated) when the delivery of drugs to the lungs by nebulisation is considered.

Anionic lipoplexes, comprising divalent cations, DNA and anionic liposomes, were optimized for high transfection efficiency and low cytotoxicity. Different molar ratios of anionic to zwitterionic lipid, 1:9 to 1:1 (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), various cations (Ca2+, Mg2+ and Na+) and different anionic lipid/DNA ratios were investigated. The optimized formulation was composed of: anionic/zwitterionic lipid molar ratio 1:4 (DOPG:DOPE); 15-20 mM Ca2+; and 15-20 microg lipid for complexation with 0.8 microg plasmid DNA. Biophysical studies (particle size analysis, gel electrophoresis, transmission electron microscopy (TEM), and confocal microscopy) were conducted to characterize the different formulations. TEM revealed structural differences between the complexed and uncomplexed lipoplexes. Gel electrophoresis confirmed the formation of anionic lipoplexes with the amount of free DNA minimized for the optimized formulation. Confocal imaging showed cellular uptake of the anionic lipoplexes. Most significantly the anionic lipoplex formulation, optimized for the highest transfection efficiency (approximately 78% in the presence of serum) exhibited the highest cell viability (approximately 93%, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) MTT assay). This was compared to Lipofectamine 2000 which had a transfection efficiency and cell viability of approximately 68% and 35%, respectively. The anionic lipoplex formulation developed here shows promise as a non-viral vector with high transfection efficiency and low cytotoxicity.

Aims: The objective of this work was to evaluate the acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin (SpHL-CDDP), after their intraperitoneal administration in male and female mice., Main Methods: After single administration of free CDDP (5,10,and 20 mg/kg) or SpHL-CDDP (7,12,30,45 and 80 mg/kg), the body weight was recorded and the LD(50) was calculated. Blood samples were collected for biochemical and hematological analysis. Kidneys, liver, spleen and bone marrow were removed to histopathological examination., Key Findings: Mice treated with high doses of free CDDP showed a greater loss of body weight and more delayed recovery time than those treated with SpHL-CDDP. The LD(50) values for SpHL-CDDP treatment for male and female mice groups were 2.7 and 3.2 fold higher, respectively, than that obtained for free CDDP. The red and white blood cells counts and quantification of hemoglobin and hematocrit presented no change upon administration of SpHL-CDDP treatment. Free CDDP treatment, however, did lead to an appearance of mild anemia and a reduction in total white blood cell counts. As regards nephrotoxicity, it was observed that free CDDP treatment caused pronounced alterations in the blood urea and creatinine levels of mice. In contrast, these parameters were slightly altered only after SpHL-CDDP treatment at a dose of 30 mg/kg. Microscopic analysis of kidneys from mice treated with SpHL-CDDP showed no morphological alteration. Concerning hepatotoxicity, no histopathological alteration was observed after both treatments., Significance: These findings reveal that SpHL-CDDP can eliminate CDDP-induced toxicity and is thus a promising candidate for intraperitoneal chemotherapy.

Various water-soluble derivatives of fullerene-C60 (C60) have been developed as detoxifiers for reactive oxygen species (ROS), whereas C60 incorporated in liposome (Lpsm) has not been reported yet. We prepared the liposome-fullerene (0.2% aqueous phase, Lpsm-Flln) which was composed of hydrogenated lecithin, glycine soja (soybean) sterols, and C60 in the weight ratio of 89.7:10:0.3, then examined the photocytotoxicity and bacterial reverse mutagenicity, as comparing with the Lpsm containing no C60. Photocytoxicity of Lpsm-Flln or Lpsm was examined using Balb/3T3 fibroblastic cells at graded doses of 0.49-1000 microg/mL under the condition of UVA- or sham-irradiation. The cells were irradiated with UVA (5 J/cm2, 320-400 nm, lambda max = 360 nm) at room temperature for 50 min. The resultant cell viability (% of control) did not decrease dose-dependently to 50% or less regardless of the UVA-irradiation. These results show that Lpsm-Flln or Lpsm does not possess photocytotoxicity to Balb/3T3 fibroblasts, and Lpsm-Flln may not exert a UVA-catalytic ROS-increasing action. A possibility for the reverse mutation by Lpsm-Flln or Lpsm was examined on four histidine-demanding strains of Salmonella typhimurium and a tryptophan-demanding strain of Escherichia coli. As for the dosages of Lpsm-Flln or Lpsm (313-5000 microg/plate), the dose-dependency of the number of reverse mutation colonies of each strain did not show a twice or more difference versus the negative control regardless of the metabolic activation, and, in contrast, marked differences for five positive controls (sodium azide, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine, and 2-aminoanthracene). The growth inhibition of bacterial strains and the deposition of Lpsm-Flln or Lpsm were not found. As a result, the bacterial reverse mutagenicity of Lpsm-Flln or Lpsm was judged to be negative under the conditions of this test. Thus, Lpsm-Flln and Lpsm may not give any significant biological toxic effects, such as photocytotoxicity and bacterial reverse mutagenicity.

This study investigated the physicochemical characteristics of cationic vesicles that were prepared from two phosphatidylcholines and three dialkyldimethylammonium bromides (DXDAB) with differing in dialkyl chain lengths, ranging from 2-C(14) to 2-C(18), by measuring particle size and zeta potential. The dependence of particle size, zeta potential and short-storage stability of mixed phosphatidylcholine/DXDAB vesicles on the chain length and composition were also elucidated. Transmission electron microscopy analysis verified that vesicles were formed as a phosphatidylcholine film to which DXDAB was added in a phosphate buffer saline (PBS, pH 7.4). Furthermore, the toxicity to the human keratinocytes (HaCaT) and squamous cell carcinomas (SCC25) cells that were incubated with these vesicles, evaluated by a cell viability assay, increased with the percentage of DXDAB that was incorporated and was inversely proportional to the chain length of DXDAB. The morphological features (round shape, chromatin condensation and apoptosis bodies) and results of flow cytometry analysis (increased sub-G(1) fraction) confirmed the induction of apoptosis in HaCaT and SCC25 cells by cationic vesicles. Apoptosis caused by cationic vesicles without the addition of any drugs was observed for the first time in HaCaT and SCC25 cells. The results of this investigation suggest that cytotoxicity is related to the zeta potential of the cationic vesicles.

To increase drug concentration in the head through intranasal administration, we have investigated the excised animal nasal mucosa permeability and nasal toxicity of the baicalin drug carrier systems, such as baicalin liposomes, beta-cyclodextrin inclusion compound, and phospholipid complex. A transport of baicalin drug carrier systems through nasal mucosa was simulated in diffusion chamber in vitro, and swine, caprine and rabbit nasal mucosa was used, the concentration of drug in the receptor was determined by HPLC. By taking the apparent permeability coefficients as evaluation standard, investigated the isolated animal nasal mucosa permeability of different baicalin drug systems was investigated for screening the best baicalin drug carrier system through nasal cavity administration. Toxicity of baicalin and its phospholipids complex on toad palate mucosal cilia movement and rats nasal mucosa long-term toxicity were studied in vivo. The apparent permeability coefficient of three kinds of baicalin drug carrier systems was better than that of baicalin (P < 0.05), and its lag-time was obviously shortened. At the same time, the apparent permeability coefficient of phospholipid complex was higher than those of other two drug carrier systems (P < 0.05). The results showed that the baicalin phospholipids complex nasal mucosa permeability was obviously superior to the other two drug systems. Baicalin phospholipids complex had no toxicity to ciliary movement, and had no irritation to rat nasal mucosa. The results show that baicalin phospholipid complex was the best baicalin drug carrier system, it could significantly enhance the permeability of baicalin across nasal mucosa, had no toxicity to nasal mucosa, and could be used for intranasal administration.

Lipid-DNA complexes (lipoplexes) are widely used, since several years, as gene carriers. However, their transfection efficiency, both in vitro and in vivo, depends, in a rather complex way, on different interconnected parameters, ranging from the chemical composition of the lipid components to the size and size distribution of the complexes and, moreover, to the composition of the suspending medium. In this paper, we have investigated the behavior of nine different commercially available transfection agents (liposomal and non-liposomal) and their lipoplexes, at different molar charge ratios and in different experimental conditions. The size and the time stability of the resulting lipoplexes were investigated by means of dynamic light scattering methods and their toxicity and transfection efficiency were assayed in vitro in a model tumor cell line (C6 rat glioma cell line). An attempt to correlate the different parameters governing the complex phenomenology observed has been made. Whereas all the formulations investigated display a low toxicity, that increases with the increase of the lipid-DNA molar charge ratio, the transfection efficiency markedly depends, besides the molar charge ratio, on the lipid composition and on the lipoplex size, in a rather correlated way. The aim of this work is to present, in a wide scenario, an example of the inter-correlation among the different parameters that influence the transfection efficiency of lipoplexes and to suggest the role exerted by the average size of the resulting aggregates in their overall effectiveness as carriers in gene therapy.

The solid phase synthesis of a library of aminoglycerol-diamine conjugate-based transfection agents having urea linkage between diverse length of diamines and various lengths of hydrophobic tails is described. These compounds were characterized and structure-activity relationships were determined for DNA binding and transfection ability when formulated as cationic liposomes. Cationic lipids with short spacer length and short hydrophobic tails bound to DNA and delivered DNA into HEK293 cells more efficient than those with longer ones. Transfection efficiency of some of the cationic liposomes was superior to that of the commercial transfection agents, Effectene, DOTAP and DC-Chol. The lipids 6Ab and 6Bb did not require the helper lipid DOPE to produce high-efficiency transfection of human cells while displaying minimal cytotoxicity. This suggests that these newly described aminoglycerol-based lipids should be very promising in liposome-mediated gene delivery and illustrate the potential of solid phase synthesis method for non-viral vector discovery.

Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both in vitro and in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tf-lipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both in vitro an in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.

An important challenge for the development of new generations of vaccines is the efficient delivery of antigens to antigen presenting cells such as dendritic cells. In the present study we compare the interaction of plain and targeted liposomes, containing mono-, di-, and tetraantennary mannosyl lipid derivatives, with human monocyte-derived immature dendritic cells (iDCs). Whereas efficient mannose receptor-mediated endocytosis by iDCs was observed for the mannosylated liposomes, in contrast, only nonspecific interaction with little uptake was observed with plain liposomes. In accordance with the clustering effect, liposomes prepared with multibranched mannosylated lipids displayed higher binding affinity for the mannose receptor than vesicles containing the monomannosylated analogs. Importantly, we have found that dimannosylated ligands present at the surface of the liposomes were as efficient as tetramannosylated ones to engage in multidentate interactions with the mannose receptor of iDCs, resulting in both cases in an effective uptake/endocytosis. This result will greatly facilitate, from a practical standpoint, the design of mannose-targeted vaccination constructs. Moreover, we showed that mannose-mediated uptake of liposomes did not result in an activation of iDCs. Altogether, our results suggest that antigen-associated targeted liposomes containing diantennary mannosylated lipids could be effective vectors for vaccines when combined with additional DC activation signals.

Objective: To investigate the toxicity of cationic liposome Lipofectamine 2000 (Lipo) in human pancreatic cancer Capan-2 cells., Methods: Capan-2 cells were cultured in the presence of Lipo at toxic concentrations, and the cell growth, apoptosis and cell cycle changes were evaluated by cell counting and flow cytometry., Results: The concentrations of both Lipo and siRNA affected the transfection efficiency. In a transfection volume of 2 ml, the presence of 5 microl Lipo resulted in slowed growth of Capan-2 cells, which was especially obvious after 3 days (P<0.001). Prolonged culture of the transfected cells caused significant increases in early apoptotic cells (P<0.05) and in the damaged or necrotic cells (P<0.001), and resulted in reduced viable cells (P<0.01); these changes became obvious after a 48-hour culture, which also increased the ratio of G(0)/G(1) phase cells (P<0.05) and decreased those of G(2)/M phase cells (P<0.01), S phase cells (P<0.01), and the late apoptotic cells (P<0.05)., Conclusion: Toxic concentrations of Lipo can affect the growth, apoptosis and cell cycles of Capan-2 cells in vitro, and this urges careful concentration selection when using Lipo for gene transfer into different cells.

Ceramides mediate antiproliferative responses, and it has been proposed that increasing the level of ceramides in cancer cells may have a therapeutic antitumor effect. However, ceramides, because of their high "packing parameter" (PP), do not form lipid assemblies that can be dispersed in a form suitable for intravenous administration. We found that nanoliposomes containing short- or medium-chain ceramides are unstable because of their very high (>1.3) PP. To overcome this major obstacle, we included the lipopolymer 2kPEG-DSPE, which reduces the additive PP. The presence of PEG-DSPE allows the formation of highly stable (>1 year) ceramide (Cer)-containing nanoliposomes suitable for systemic administration. Using tumor cell lines, we found that the ceramide cytotoxicity was not impaired by their inclusion in nanoliposomes. The use of 14C-labeled ceramides shows that the C6Cer, but not C16Cer, was transferred from the nanoliposomes to the cells and metabolized efficiently. The difference between the two ceramides is related to the large difference between their critical aggregation concentration and was correlated with the much higher cytotoxity of liposomal C6Cer. The activity of 2kPEG-DSPE as a steric stabilizer (as previously shown for Doxil) was also confirmed for C6Cer-containing nanoliposomes. The 2kPEG-DSPE lipopolymer significantly reduced the desorption rate of the ceramide from the liposome bilayer, thereby allowing liposomes containing C6Cer to reach the tumor site and to demonstrate therapeutic efficacy.

Clinical success of many therapies is impaired by dose limiting toxicities. Nanoscale particle-based drug delivery systems such as liposomes show unique pharmacokinetic properties and improved toxicity profiles. Liposomes accumulate in tumor tissue, but only a small fraction of a total dose reaches the target site. The overwhelming amount of a given dose is needed only to build up a diffusion gradient for effective accumulation at the target site. In order to find a way to detoxify this predominant fraction after accumulation is completed, the different separation principles used for the apheresis of lipoproteins were evaluated for the extracorporeal elimination of liposomes. Appropriate radiolabeled model liposomes were prepared by extrusion. Separation efficacy, leakage of liposomal content and influence of plasma contact were measured. Membranes with pore sizes between 25 and 400 nm were used to investigate filtration properties of liposomes. Liposomes were precipitated by adding heparin and Ca(2+). Adsorption chromatography was investigated using dextran sulfate, heparin sepharose and functionalized polyacrylamide beads. Membrane filtration allowed the elimination of various liposomes, while precipitation and adsorption were only useful for positively charged liposomes. Leakage of liposomal content was not induced by adsorption, but precipitation induced leakage. Leakage during filtration was dependent on liposomal membrane lipids. Plasma contact reduced precipitation and adsorption efficacy of positively charged liposomes, while filtration properties of liposomes remained unchanged. For extracorporeal elimination of liposomal drug delivery systems, filtration-based techniques are presumably more convenient and versatile than precipitation- or adsorption-based apheresis technologies.

A novel bimodal fluorescent and paramagnetic liposome is described for cellular labeling. In this study, we show the synthesis of a novel gadolinium lipid, Gd.DOTA.DSA, designed for liposomal cell labeling and tumor imaging. Liposome formulations consisting of this lipid were optimized in order to allow for maximum cellular entry, and the optimized formulation was used to label HeLa cells in vitro. The efficiency of this novel bimodal Gd-liposome formulation for cell labeling was demonstrated using both fluorescence microscopy and magnetic resonance imaging (MRI). The uptake of Gd-liposomes into cells induced a marked reduction in their MRI T 1 relaxation times. Fluorescence microscopy provided concomitant proof of uptake and revealed liposome internalization into the cell cytosol. The optimized formulation was also found to exhibit minimal cytotoxicity and was shown to have capacity for plasmid DNA (pDNA) transfection. A further second novel neutral bimodal Gd-liposome is described for the labeling of xenograft tumors in vivo utilizing the enhanced permeation and retention effect (EPR). Balb/c nude mice were inoculated with IGROV-1 cells, and the resulting tumor was imaged by MRI using these in vivo Gd-liposomes formulated with low charge and a poly(ethylene glycol) (PEG) calyx for long systemic circulation. These Gd-liposomes which were less than 100 nm in size were shown to accumulate in tumor tissue by MRI, and this was also verified by fluorescence microscopy of histology samples. Our in vivo tumor imaging results demonstrate the effectiveness of MRI to observe passive targeting of long-term circulating liposomes to tumors in real time, and allow for MRI directed therapy, wherein the delivery of therapeutic genes and drugs to tumor sites can be monitored while therapeutic effects on tumor mass and/or size may be simultaneously observed, quantitated, and correlated.

Small unilamellar amphotericin B liposomes can reduce the toxicity of amphotericin B. In this study, we compared the physical, antifungal, pharmocokinetic, and toxic properties of two liposomal amphotericin B products, AmBisome and Anfogen, that have the same chemical composition but are manufactured differently. In vitro tests included determinations of the MICs and the concentrations causing the release of 50% of the intracellular potassium from red blood cells (K50 values) to assess toxicity. The 50% lethal dose (LD50) was evaluated by using uninfected C57BL/6 mice and single intravenous (i.v.) doses of 1 to 100 mg/kg of body weight. Multiple i.v. dosing over 18 days was performed with 0.5, 1.0, or 5.0 mg of Anfogen/kg or 1.0, 5.0, or 25 mg of AmBisome/kg to evaluate chronic toxicity. DBA/2 mice were infected intranasally with 2.5 x 10(6) Aspergillus fumigatus conidia, treated for 3 or 4 days with 3.0, 5.0, or 7.5 mg of Anfogen/kg or 3, 5, 7.5, or 15 mg of AmBisome/kg, and evaluated to assess the toxicity of the drugs to the kidneys (by measurement of blood urea nitrogen and creatinine levels and histopathology) and the drug efficacy. The median particle size was 77.8 nm for AmBisome and 111.5 nm for Anfogen. In vitro K(50) values were significantly lower for Anfogen (0.9 mug/ml) than for AmBisome (20 microg/ml), and the LD50 of AmBisome was >100 mg/kg, versus 10 mg of Anfogen/kg. There was significant renal tubular necrosis in uninfected and infected mice given Anfogen but no tubular necrosis in AmBisome-treated mice. AmBisome at 7.5 or 15 mg/kg was also more efficacious than 7.5 mg of Anfogen/kg for the treatment of pulmonary aspergillosis, based on survival and weight loss data and numbers of CFU per gram of lung. In conclusion, the efficacy and toxicity of these two liposomal amphotericin B products were significantly different, and thus, the products were not comparable.

Cationic liposomes with high stability and low cytotoxicity for gene therapy have been developed. Luciferase plasmid DNA (pLuc) was used as a model gene. The empty liposomes and niosomes were prepared by freeze dried empty liposomes (FDEL) method. The entrapment of pLuc in the liposomes was by reconstitution of the lyophilized dried vesicles with the plasmid solution. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM) and cryo-TEM. Cytotoxicity of the vesicular formulations was investigated on mouse melanoma cell lines (B16F10) by MTT assay. Cationic lposomes and niosomes containing the cationic lipid DDAB were less cytotoxic than other bilayer vesicular formulations. The pLuc entrapped in the cationic DPPC/Chol/DDAB liposomes (at 1:1:1 molar ratio) exhibited higher stability than other vesicular formulations and the pLuc in solution when stored at 4, 30 and 50 degrees C for 8 weeks. The entrapment efficiency determined by gel electrophoresis and gel documentation of the pLuc in this liposomal formulation was 100%. Luciferase gene expression of pLuc-loaded in cationic liposomes (lipoplexes) in HeLa cell lines was evaluated from luciferase activity determined by a luminometer at 24 and 48 h incubation. Percentages of cell proliferation of the lipoplexes on HeLa cell line at 24 and 48 h incubation were evaluated by the WST-1 assay. When the amount of DPPC or cholesterol was increased in the lipoplexes, the higher amount of DDAB was needed to protect pLuc from enzymatic degradation. However, DPPC and cholesterol exceeded 33 and 50% mol, respectively gave no gene expression. The DPPC/Chol/DDAB (at 1:1:1 molar ratio) lipoplex has demonstrated moderately lower luciferase gene expression and low cytoxicity. This lipoplex with the DDAB/pLuc weight ratio of 14:1 was the most desirable formulation for gene therapy because of its high stability, high luciferase gene expression and low cytotoxicity.

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. The development of efficient and safe gene transfer systems could be one of the most important factors for successful gene therapy. In the present study, an amphiphilic compound, polyethylenimine (PEI, MW 800)-cholesterol (PEI 800-Chol), firstly designed to modify the surface of liposomes, was synthesized. Polycation liposomes (PCLs) composed of soybean phospholipids (SPL), cholesterol (Chol) and PEI 800-Chol were prepared using film hydration method. The mean particle size of the PCLs was 133.0 nm and the zeta potential was 50.1+/-2.6 mV. Due to the PEI anchored onto the surface of liposomes, higher buffering capacity of PCLs was observed, indicating the potential for buffering in the acidic pH environment of the endosomes. Compared to Lipofectamine 2000, PCLs have equivalent transfection efficiency with significantly low cytotoxicity. Interestingly, the transfection activity of PCLs was not influenced in the presence of serum. Furthermore, we constructed another PCL composed of PEI 800-Chol and DOPE, and transfection efficiency increased notably. In conclusion, the PCLs described in this study have high transfection efficiency with low cytotoxicity, as well as the protection ability from serum, which suggests PCLs would be a potential non-viral gene delivery system.

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.

Five cholesterol-based gemini cationic lipids, which differ in the length of the spacer [-(CH2)n-] chain between the head groups, have been synthesized. These lipids are useful as nonviral gene delivery agents, and all cholesterol-based gemini lipids (2a-2e) are better transfecting agents than their monomeric lipid counterpart 1. Transfection efficiency of all the gemini lipids except lipid 2a [-(CH2)3-] was maintained even when the serum was present during the transfection conditions as compared to the monomeric lipid 1, with which a dramatic decrease in transfection efficiency was observed. With the increase in spacer chain length from propanediyl [-(CH2)3-] to pentanediyl [-(CH2)5-], transfection efficiency increased both in the absence and presence of serum. However, transfection efficiency decreased with further increase in the length from the pentanediyl [-(CH2)5-] to the dodecanediyl [-(CH2)12-] spacer. Among these gemini lipids 2c showed the highest transfection activity, which was also greater than that of the commercially available formulation.

Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.

The influence of various formulation factors on the in vitro cellular toxicity of liposomes on human buccal cells (TR146), were studied by using the concept of statistical experimental design and multivariate evaluation. The factors investigated were the type of main phospholipid (egg-PC, DMPC, DPPC), lipid concentration, the type of charge, liposome size, and amount and nature of the charged component (diacyl-PA, diacyl-PG, diacyl-PS, stearylamine (SA), diacyl-TAP) in the liposomes. Both full factorial design and D-optimal designs were created. Several significant main factors and interactions were revealed. Positively charged liposomes were shown to be toxic. The toxicity of negatively charged liposomes was relatively low. Diacyl-TAP was less toxic than SA, and DPPC was less toxic than DMPC. Low level of positively charged component was favourable and essential when using egg-PC as the main lipid. The amount of negatively charged component, the liposome size, and the total lipid concentration did not affect the toxicity within the experimental room. DPPC appeared to be a good candidate when formulating both positively and negatively charged liposomes with low cellular toxicity. The concept of statistical experimental design and multivariate evaluation was shown to be a useful approach in cell toxicity screening studies.

Gene therapy, as a promising therapeutics to treat genetic or acquired diseases, has achieved exciting development in the past two decades. Appropriate gene vectors can be crucial for gene transfer. Cationic lipids and polymers, the most important non-viral vectors, have many advantages over viral ones as non-immunogenic, easy to produce and not oncogenic. They hold the promise to replace viral vectors to be used in clinic. However, the toxicity is still an obstacle to the application of non-viral vectors to gene therapy. For overcoming the problem, many new cationic compounds have been developed. This article provides a review with respect to toxicity of cationic lipids and polymers in gene delivery. We evaluate the structural features of cationic compounds and summarize the relationship of toxicity and structure and hope to provide available suggestions on the development of these cationic compounds.

Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.

Liposome formulations of camptothecins have been actively pursued because of the potential for significant pharmacologic advantages from successful drug delivery of this important class of anticancer drugs. We describe nanoliposomal CPT-11, a novel nanoparticle/liposome construct containing CPT-11 (irinotecan) with unprecedented drug loading efficiency and in vivo drug retention. Using a modified gradient loading method featuring a sterically hindered amine with highly charged, multivalent anionic trapping agents, either polymeric (polyphosphate) or nonpolymeric (sucrose octasulfate), liposomes were capable of entrapping CPT-11 at extremely high drug-to-lipid ratios (>800 g CPT-11/mol phospholipid) and retaining encapsulated drug in vivo with a half-life of drug release in the circulation of 56.8 hours. CPT-11 was also protected from hydrolysis to the inactive carboxylate form and from metabolic conversion to SN-38 while circulating. The maximum tolerated dose in normal mice was determined to be 80 mg/kg for free CPT-11 and >320 mg/kg for nanoliposomal CPT-11. Nanoliposomal CPT-11 showed markedly superior efficacy when compared with free CPT-11 in human breast (BT474) and colon (HT29) cancer xenograft models. This study shows that intraliposomal stabilization of CPT-11 using a polymeric or highly charged, nonpolymeric polyanionic trapping agent results in a markedly active antitumor agent with low toxicity.

We hypothesized that combining convection-enhanced delivery (CED) with a novel, highly stable nanoparticle/liposome containing CPT-11 (nanoliposomal CPT-11) would provide a dual drug delivery strategy for brain tumor treatment. Following CED in rat brains, tissue retention of nanoliposomal CPT-11 was greatly prolonged, with >20% injected dose remaining at 12 days for all doses. Tissue residence was dose dependent, with doses of 60 microg (3 mg/mL), 0.8 mg (40 mg/mL), and 1.6 mg (80 mg/mL) resulting in tissue half-life (t(1/2)) of 6.7, 10.7, and 19.7 days, respectively. In contrast, CED of free CPT-11 resulted in rapid drug clearance (tissue t(1/2) = 0.3 day). At equivalent CED doses, nanoliposomal CPT-11 increased area under the time-concentration curve by 25-fold and tissue t(1/2) by 22-fold over free CPT-11; CED in intracranial U87 glioma xenografts showed even longer tumor retention (tissue t(1/2) = 43 days). Plasma levels were undetectable following CED of nanoliposomal CPT-11. Importantly, prolonged exposure to nanoliposomal CPT-11 resulted in no measurable central nervous system (CNS) toxicity at any dose tested (0.06-1.6 mg/rat), whereas CED of free CPT-11 induced severe CNS toxicity at 0.4 mg/rat. In the intracranial U87 glioma xenograft model, a single CED infusion of nanoliposomal CPT-11 at 1.6 mg resulted in significantly improved median survival (>100 days) compared with CED of control liposomes (19.5 days; P = 4.9 x 10(-5)) or free drug (28.5 days; P = 0.011). We conclude that CED of nanoliposomal CPT-11 greatly prolonged tissue residence while also substantially reducing toxicity, resulting in a highly effective treatment strategy in preclinical brain tumor models.

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as beta-sitosterol beta-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.

A new type of boron-rich, DSPC-free, unilamellar liposomes was formed using the novel dual-chain, ionic, nido-carborane lipid, K[nido-7-(C16H33OCH2)2CHOCH2-7,8-C2B9H11] (DAC-16), and cholesterol for encapsulation of an aqueous buffer core. Since DSPC was not necessary for the formation of stable DAC-16 liposomes, the boron concentration of these vesicles was increased dramatically to approximately 8.8 wt % in the dry lipid; these liposomes had a high bilayer boron incorporation efficiency of 98%. DSPC-free liposomes exhibited a size distribution pattern of 40-60 nm, which was in the range normally associated with selective tumor uptake. This size distribution was maintained throughout storage at room temperature for several months. Additionally, optimized liposome formulations incorporating DAC-16, DSPC, and cholesterol were identified having stable size distribution patterns after storage for more than two months at a variety of temperatures. Although animal studies indicate that DAC-16 liposomes are toxic, this new ionic nido-carborane lipid allows the formation of liposomes of high boron content for in vitro applications that require the delivery of large amounts of boron.

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.

Implementation of the high-throughput microarray gene expression profiling technology towards "toxicogenomics" has advanced identification process for safer drugs in the century of 'omics' technology. Applying such technology, in fact, to identify mechanisms for cellular toxicity can provide a means to clarify safety liabilities early in the drug discovery and developments process. The underlying principle in gene therapy is primarily targeting a specific gene (e.g., for silencing). Hence, massive efforts have been devoted to validate the gene-based therapeutics, regardless of toxicogenomics potential of delivery systems. Of the gene delivery systems, viral and non-viral vectors, as two main paradigms, have so far been widely used for delivering of the genome-based therapeutics such as oligonucleotide, small interfering RNA and DNA. However, the use of viral vectors was narrowed due to the safety concerns. Non-viral vectors were utilized as safer alternatives for gene delivery in vitro and ex-vivo; though their success for in vivo gene therapy has been limited due to low efficiency and safety issues. Fundamental principle for gene therapy is to deliver gene-based therapeutics into target cells for specific gene targeting ideally with minimal cellular toxicity. Until now, few works have been conducted about geno-compatibility of delivery systems itself, including cationic lipid-based nanosystems. Inadvertent toxicogenomic impact of gene delivery systems (e.g., cationic lipids) may intrinsically affect the outcome of gene therapy, where often only a single desired genetic change is sought. Further, there exists a possibility that gene changes induced by the lipid delivery system itself could exacerbate, attenuate or even mask the desired effects of the gene-based therapeutics. This review will focus on toxicogenomics impact of the cationic lipid-based formulations for gene therapy.

C/EBP beta (CCAAT/enhancer-binding protein beta) is an important transcription factor involved in cellular proliferation and differentiation. Overexpression of the full-length C/EBP beta protein results in cellular growth arrest and apoptosis. Using a nonviral liposome as carrier, we delivered the full-length C/EBP beta expression plasmid, pCN, into nude mice bearing CW-2 human colon cancer tumors via tail vein. Southern blots revealed that the major organs and tumors were transfected. Experimental gene therapy showed that a strong suppression of tumor growth was observed in the pCN-treated mice, and such suppression was due to the overexpression of C/EBP beta, leading to the increased apoptosis in tumors of pCN-treated mice. No apparent toxic effects of pCN/liposome complex were observed in the animals. Thus, C/EBP beta has tumor suppression effect in vivo and may be used in gene therapy for cancers.

Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

Inflammatory toxicity represents a typical toxicity associated with systemic administration of cationic liposome/DNA complex (lipoplex). Collected information indicates that the lipoplex gene delivery system mediates an uptake of plasmid DNA by the liver, mainly by Kupffer cells, in which a large amount of cytokine is produced. Therefore, many efforts have been made to overcome this problem. Previous reports by our laboratory demonstrated that sequential injection of cationic liposome and DNA could dramatically decrease the toxicity. In comparison with lipoplex injection, this method significantly suppresses the uptake of DNA by the liver. Opsonization effect in the stimulation of Kupffer cell uptake is proposed as an explanation for the differences in the pharmacokinetic properties of plasmid DNA after lipoplex injection and sequential injection. In this review, we cover the current understanding of the mechanisms underlying inflammatory toxicity and the several attempts to overcome this toxicity. The mechanism related to the pharmacokinetic properties of the lipoplex is focused on here for discussion.

A series of thiocholesterol-based cationic lipids (TCL) has been designed and synthesized by the attachment of thiocholesterol to a cationic amine via a disulfide bond. TCL can be incorporated into liposomes and used to package DNA into a lipoplex, thereby protecting it from DNase digestion. DNA is rapidly released from the complex in the presence of low concentrations of reducing agents. The lipoplex mediated efficient transfection activity and had low cytotoxicity. To improve the biocompatibility of the cationic lipoplex, TCL were used as a component in the assembly of a nanolipoparticle (NLP). The particle surface was subsequently modified by disulfide exchange to replace the cationic group with a negatively charged (glutathione) or zwitterionic (cysteine) reducing agent. A cell-binding ligand (TAT peptide, sequence GRKKRRQRRRGYG) was then incorporated onto the particle surface to enhance the particle-cell recognition. The sequentially assembled cell-binding NLP with a zwitterionic surface gave a larger transfection yield than the cationic NLP at all concentrations tested. At low DNA concentrations, the enhancement was 80-fold. The disulfide cationic lipids and the sequential assembly strategy enable one to tailor the surface charge, hydrophilicity, and recognition elements of a nanosized gene carrier. This results in increased gene transfer activity in a biocompatible particle.

Gene therapy has been deemed the medicine of the future due to its potential to treat many types of diseases. However, many obstacles remain before gene delivery is optimized to specific target cells. Over the last several decades, many approaches to gene delivery have been closely examined. By understanding the factors that determine the efficiency of gene uptake and expression as well as those that influence the toxicity of the vector, we are better able to develop new vector systems. This chapter will provide a brief overview of recent advances in gene delivery, specifically on the development of novel non-viral vectors. The following chapters will provide additional details regarding the evolution of non-viral gene delivery systems.

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.

For the past 15 years cationic liposomes have routinely been utilised for the delivery of nucleic acids such as plasmids and oligodeoxynucleotides to cells in culture and in vivo. These reagents are commercially available or are formulated inhouse. However, particularly in cultured cells, toxicity remains a significant problem, and this is confirmed by several in vivo findings. In addition, these complexes exhibit an immunostimulation effect, a phenomenon that may either be harmful or beneficial. Furthermore, lipoplexes have been recently found to have a certain degree of selectivity for dividing vascular endothelial cells. The development of cationic lipids that are safe to use especially in vivo and possess enhanced transfection capabilities is an ongoing process. More research is needed to understand the basic biology behind lipofection, first at the cellular level, then at the multicellular (whole organism) level.

Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43-58) and HIV Tat (aa 47-57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 +/- 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine-(5'- 5')-3'-C-ethinylcytidine showed a reduction of the IC50 by 70% on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.

The transfection efficiency of liposome-based DNA formulations was studied in different salmonid cell lines of hepatocyte and macrophage origin. Parallel assessment of cell viability was carried out to define the balance between transfection efficiency and toxicity. For all cell lines, transfection efficiency varied with the lipoplex charge ratio and the amount of DNA added to the liposomes. The hepatocyte-derived cell line was most readily transfected while lower transfection efficiency was observed for the macrophage cell lines. The cationic liposomes showed a dose-dependent toxicity and were found to be most toxic for cells of macrophage origin. This was in line with the observation that higher amounts of lipids were associated with the cells of macrophage origin than the hepatocytes. Complexing DNA with the liposomes reduced the toxicity for all three cell lines, most markedly, however, for macrophage cell lines. The differences in the transfection and toxicity patterns between the cell lines are probably caused by differences in membrane composition as well as differences in phagocytic activity and processing of the liposomes/lipoplexes.

The toxicity of 2 new synthetic lipids, 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol, GDO-12 (lipid 1) and 1,2-distearoyl-rac-glycerol-3-dodecaethylene glycol, GDS-12 (lipid 2) has been evaluated in acute and subchronic toxicity studies. Acute oral toxicity studies in male and female rats documented no deaths or treatment-related signs at high doses. The lipids were individually administered (by gavage) to male and female Sprague-Dawley rats at concentrations of 250, 500, and 1000 mg/Kg bodyweight for 28 days. All animals survived the duration of the study, with no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathological findings. These studies establish that both GDO-12 (lipid 1) and GDS-12 (lipid 2) are nontoxic in rats following oral administration. The no-observed-adverse-effect level ranged between 250 mg/Kg and 1000 mg/Kg following oral administration.

Background: Control of the structure and physicochemical properties of DNA complexed with nonviral vectors is essential for efficient biodistribution and gene delivery to cells. Cationic liposomes interact with DNA giving transfection competent but large and heterogeneous aggregates. On the other hand, cationic detergents condense DNA into small homogeneous but reversible complexes inefficient for transfection., Methods: In order to combine the favorable features of both vectors, ternary complexes were prepared by adding cationic liposomes to plasmid DNA condensed by cationic detergents. The structure and physicochemical properties of these complexes were investigated by electron microscopy, quasi-elastic light scattering, gel electrophoresis and fluorescence techniques. These data were then correlated with the transfection efficiency and intracellular trafficking of the ternary complexes determined by luciferase gene expression and confocal microscopy, respectively., Results: The ternary complexes were found to form small, homogeneous, globular, stable and positively charged particles with a highly dense and packed lamellar internal structure differing from the multilamellar structure (L(alpha)(C)) of the corresponding lipoplexes. In the presence of serum, the ternary complexes were more efficiently internalized into cells, less toxic and showed 20-fold higher transfection efficiency than lipoplexes., Conclusions: This study showed that small, monodisperse and highly stable complexes could be obtained by precompaction of DNA with cetyltrimethylammonium bromide, followed by addition of cationic lipids. The higher efficiency of the ternary complexes with respect to their corresponding lipoplexes was related to their internal structure which prevents their dissociation by serum proteins and allows efficient internalization in the target cells., (Copyright 2004 John Wiley & Sons, Ltd.)

Although liposomes have been used as a vehicle for delivery of therapeutic agents in oncology, their efficacy in targeting brain tumors has been limited due to poor penetration through the blood-brain barrier. Because convection-enhanced delivery (CED) of liposomes may improve the therapeutic index for targeting brain tumors, we conducted a three-stage study: stage 1 established the feasibility of using in vivo magnetic resonance imaging (MRI) to confirm adequate liposomal distribution within targeted regions in normal rat brain. Liposomes colabeled with gadolinium (Gd) and a fluorescent indicator, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid [DiI-DS; formally DiIC(18)(3)-DS], were administered by CED into striatal regions. The minimum concentration of Gd needed for monitoring, correlation of infused volume with distribution volume, clearance of infused liposome containing Gd and DiI-DS (Lip/Gd/DiI-DS), and potential local toxicity were evaluated. After determination of adequate conditions for MRI detection in normal brain, stage 2 evaluated the feasibility of in vivo MRI monitoring of liposomal distribution in C6 and 9L-2 rat glioma models. In both models, the distribution of Lip/Gd/DiI-DS covering the tumor mass was well defined and monitored with MRI. Stage 3 was designed to develop a clinically relevant treatment strategy in the 9L-2 model by infusing liposome containing Gd (Lip/Gd), prepared in the same size as Lip/Gd/DiI-DS, with Doxil, a liposomal drug of similar size used to treat several cancers. MRI detection of Lip/Gd coadministered with Doxil provided optimum CED parameters for complete coverage of 9L-2 tumors. By permitting in vivo monitoring of therapeutic distribution in brain tumors, this technique optimizes local drug delivery and may provide a basis for clinical applications in the treatment of malignant glioma.

Successful cancer gene therapy depends on the development of non-toxic, efficient, tumor cell- specific systemic gene delivery systems. Our laboratory has developed a systemically administered, ligand-liposome complex that can effectively and preferentially deliver its therapeutic payload to both primary and metastatic tumors. To further improve the transfection efficiency of this targeting complex, a synthetic pH-sensitive histidylated oligolysine K[K(H)KKK]5-K(H)KKC (HoKC), designed to aid in endosomal escape and condensation of DNA, was included in the complex. The presence of HoKC increased the in vitro transfection efficiency over that of the original complex. Moreover, no increase in cytotoxicity was observed due to the presence of the HoKC peptide. In a DU145 human prostate cancer xenograft tumor model in athymic nude mice, inclusion of the HoKC peptide did not interfere with the tumor targeting specificity of the i.v. administered ligand/liposome/DNA complex. Most importantly, the level of transgene expression was significantly elevated in the tumors, but not in the normal tissue in those animals receiving the complex incorporating HoKC. The in vivo enhancement of transfection efficiency by this modified gene delivery vehicle could lead to a reduction in the number of administrations required for antitumor efficacy.

Aim: To develop a hepatocyte targeting pH-sensitive liposome for drug delivery based on active targeting technology mediated by asialoglycoprotein receptors., Methods: Four types of targeting molecules with galactose residue were synthesized and mixed with pH-sensitive lipids DC-chol/DOPE to prepare liposome with integrated property of hepatocyte specificity and pH sensitivity. Liposome 18-gal was selected with the best transfection activity through cellular uptake experiment. Property analysis was made through experiments of competitive inhibition of receptors, red blood cell hemolysis, in vitro cytotoxicity test by MTS assay and mediation of inhibitory effects of antisense phosphorothioate ODN on gene expression, etc., Results: Liposome 18-gal had the desired properties of hepatocyte specificity, pH sensitivity, low cytotoxicity, and high transfection efficiency., Conclusion: Liposome 18-gal can be further developed as a potential hepatocyte- targeting delivery system.

The taxanes, paclitaxel and docetaxel, are anticancer agents used in clinical trials against ovarian carcinoma, breast, lung and head/neck cancer. Paclitaxel, very insoluble in water, is generally formulated using Cremophor EL. Docetaxel, more soluble in water, is formulated using Tween 80 and ethanol. Tween 80, albeit less toxic than Cremophor EL, may be responsible of some toxic effects. To eliminate these vehicles and improve the drug's antitumor efficacy, taxanes have been incorporated in liposomes. We compared formulation, stability, biodistribution and pharmacokinetics of docetaxel in conventional and PEGylated liposomes. Of the several formulations examined, docetaxel-liposomes composed of ePC/PG/CHOL 9:1:2 and ePC/PG/DSPE-PEG2000/CHOL 9:1:2:0.7 were the most effective. Both conventional and PEGylated docetaxel-liposomes were stable at 4 degrees C after 15 days, whereas in the presence of serum at 37 degrees C they were less stable. The IC50 values of docetaxel-liposomes, evaluated on HT-29 and Igrov1 cell lines, remained very high. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of [14C]docetaxel, formulated in Tween 80 or in 3H-labeled conventional or PEGylated liposomes. The t(1/2)beta, which was low for docetaxel (52.3 min), rose to 260 min for conventional docetaxel-liposomes and to 665 min for PEGylated docetaxel liposomes. Biodistribution studies confirmed the pharmacokinetics.