lipofectamine and 1-2-dioleoyloxy-3-(trimethylammonium)propane

Functionalized single-walled carbon nanotubes (SWNT) with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as novel and more convenient carriers of small interfering RNA (siRNA).. To utilize the unique capability of SWNT to be easily modified by functional groups and readily internalized by mammalian cells to bind, condense, stabilize siRNA and enhance its transfection efficiency.. After SWNT were non-covalently functionalized by cationic DOTAP (SWNT-DOTAP), siRNA interacted with SWNT-DOTAP via static electricity (SWNT-DOTAP/siRNA). Subsequently, the size, zeta potential and morphology of SWNT-DOTAP/siRNA were analyzed. The optimal compression ratio and stability of siRNA were assessed by agarose gel electrophoresis. Furthermore, in prostate carcinoma PC-3 cells, RT-PCR, flow cytometry and sulforhodamine B assays were used to evaluate the silencing activity, transfection efficiency and cell proliferation, respectively.. The characteristics of SWNT-DOTAP, i.e. an average size of 194.49 nm, a zeta potential of 45.16 mV and lower cytotoxicity than Lipofectamine 2000, indicated that this vector was suitable for siRNA delivery. Moreover, after interaction with SWNT-DOTAP, siRNA of human telomerase reverse transcriptase was bound, condensed and stabilized. In PC-3 cells, SWNT-DOTAP/siRNA exhibited 82.6% silencing activity and 92% transfection efficiency. Furthermore, the complexes inhibited cell proliferation by 42.1%.. SWNT-DOTAP may be a promising siRNA delivery vector for gene-based therapeutic applications in cancer.

Topics: Cations; Cell Line, Tumor; Cell Proliferation; Drug Carriers; Fatty Acids, Monounsaturated; Gene Silencing; Gene Transfer Techniques; Genetic Vectors; Humans; Lipids; Nanotubes, Carbon; Quaternary Ammonium Compounds; RNA, Small Interfering; Transfection

The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency) a system was hypothesized in which DOTAP/phosphatidyl choline (PC) lipoplexes were electrostatically coated with anionic poly(acrylic acid) (PAA) and cationic polyethylenimine (PEI) alternatively to finally shape a robust structure PEI-PAA-DOTAP/PC-lipoplexes (nanoplexes). The nanoplexes were found to have size of 242.6 ± 9.4 nm and zeta potential of +23.1 ± 1.5 mV. Following development nanoplexes were evaluated for cellular uptake, nuclear colocalization, transfection efficiency, and cellular toxicity in MCF-7, HeLa, and HEK-293 cell lines. In support of our hypothesis nanoplexes exhibited higher uptake and nuclear colocalization in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, and PEI polyplexes. Nanoplexes also exhibited 50-80, 11-12, 6-7, and 5-6 fold higher transfection efficiency in comparison with DOTAP/PC-lipoplexes, DOTAP/DOPE-lipoplexes, PEI-polyplexes, and lipofectamine, respectively, and significantly lower toxicity in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, PEI polyplexes, and commercial lipofectamine.

Topics: Acrylic Resins; Cell Line; Cell Line, Tumor; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Vectors; HeLa Cells; Humans; Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Polyethyleneimine; Polymers; Quaternary Ammonium Compounds; Transfection

Messenger RNA encoding luciferase (mLUC) was complexed to the cationic lipids Lipofectamine or DOTAP/DOPE, and to the cationic polymer linear poly(ethyleneimine) (linPEI). The complexes were incubated with HeLa cells and luciferase expression was assessed. The type of non-viral carrier used determined the extent and duration of protein expression. Maximal duration of mRNA expression was about 9 days for Lipofectamine complexes, i.e. not very much shorter than with pDNA polyplexes. Interestingly, luciferase activity was already detected 30 min after adding the mRNA complexes to the cells, independent on the type of carrier. We also assessed the proportion of cells that become transfected by means of transfection with an mRNA encoding GFP. For both cationic lipids transfection with mRNA yielded a substantially larger fraction of transfected cells (more than 80%) than transfection with pDNA (40%). In addition we tested the carriers for their ability to mediate delivery of mRNA encoding CXCR4 into mesenchymal stem cells. The fraction of CXCR4-positive cells obtained with the mRNA-cationic lipid complexes was around 80%, as compared to 40% for the linPEI polyplexes. Our results demonstrate that the advantage of the use of mRNA over that of pDNA may under certain conditions outweigh the disadvantage of the somewhat shorter expression period.

Topics: Animals; Cell Survival; Fatty Acids, Monounsaturated; Female; Green Fluorescent Proteins; HeLa Cells; Humans; Kinetics; Lipids; Luciferases; Mesenchymal Stem Cells; Phosphatidylethanolamines; Plasmids; Polyamines; Polyelectrolytes; Polyethyleneimine; Quaternary Ammonium Compounds; Receptors, CXCR4; RNA, Messenger; Transfection; Uterine Cervical Neoplasms

Interleukin-18 (IL-18) is known to reduce melanoma lung metastases through various mechanisms. For the delivery of IL-18 gene into the lung, three different cationic emulsions as non-viral vectors were formulated using the same components of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), and Tween 80 with distinct oils. By using the small particle size of physicochemically stable E3, the complex of E3/plasmid DNA encoding IL-18 (16:2.5, w/w) was transfected into lung cancer cells, and the amount of plasmid DNA transferred and the expression of both mRNA and protein for IL-18 were measured. When compared with Lipofectamine/DNA complexes, an E3/DNA complex was less toxic and induced a comparable cellular level of plasmid DNA and expression levels of both mRNA and protein for IL-18. After injecting E3/DNA complexes into mice, the distribution of plasmid DNA was the highest in the lung and the liver. Especially, the administration of E3/DNA complexes induced a more rapid and prolonged distribution of plasmid DNA encoding IL-18 into the lung than that of Lipofectamine/DNA ones. These data demonstrated that cationic emulsion E3 containing castor oil could be useful for a delivery of IL-18 gene targeting the lung as well as the liver without an additional homing device, implying a potential IL-18 delivery system for the treatment of lung cancer.

Topics: Animals; Castor Oil; Cell Line, Tumor; DNA; DNA Adducts; Drug Stability; Emulsifying Agents; Emulsions; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Injections, Intravenous; Interleukin-18; Lipids; Mice; Mice, Inbred BALB C; Olive Oil; Phosphatidylethanolamines; Plant Oils; Plasmids; Polysorbates; Quaternary Ammonium Compounds; RNA, Messenger; Soybean Oil; Swine

Human 16K PRL (16K hPRL) is a potent inhibitor of angiogenesis both in vitro and in vivo. It has been shown to prevent tumor growth in three xenograft mouse models. Here we have used a gene transfer method based on cationic liposomes to produce 16K hPRL and demonstrate that 16K hPRL inhibits tumor growth in a subcutaneous B16F10 mouse melanoma model. Computer-assisted image analysis shows that 16K hPRL treatment results in the reduction of tumor vessel length and width, leading to a 57% reduction in average vessel size. We thus show, for the first time, that administration of the 16K hPRL gene complexed to cationic liposomes is effective to maintain antiangiogenic activities of 16K hPRL level.

Topics: Angiogenesis Inhibitors; Animals; Cations; Cell Line; Cholesterol; DNA, Recombinant; Drug Screening Assays, Antitumor; Fatty Acids, Monounsaturated; Female; Genetic Therapy; Humans; Kidney; Lipids; Liposomes; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Prolactin; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Transfection

In order to study the important factors involved in cationic liposome-mediated gene transfer, Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies. Different properties of the two reagents were analyzed and compared by DNA arrearage assay and MTT assay. Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell. However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell. On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition. Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.

Topics: Cell Line, Tumor; Cell Survival; DNA; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Humans; Lipids; Quaternary Ammonium Compounds; Transfection

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.

Topics: Animals; Brain; Cell Nucleus; Cell Survival; Cells, Cultured; Central Nervous System; Cholesterol; Cytoplasm; Drug Delivery Systems; Fatty Acids, Monounsaturated; Gene Expression; Gene Silencing; JNK Mitogen-Activated Protein Kinases; Lipids; Liposomes; Luciferases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence; Neurons; Quaternary Ammonium Compounds; RNA, Small Interfering; Transfection; Transferrin

We have reported earlier that a combination of low-molecular weight polyethylenimines (PEIs) with the cationic liposome, Dosper, results in a synergistic increase in the transfection efficiency. Now we have investigated whether this synergism is a general mechanism seen with other transfection reagents as well. Therefore, we have combined the low-molecular weight PEIs (MW 700 and 2000) with Dotap (a monocationic liposome), Lipofectamine (a combination of neutral and polycationic liposome), and Superfect (a dendrimer). The highest synergism was achieved with Lipofectamine and PEIs in the SMC cells, or with Dotap and PEIs in the C6 cells. Superfect did not induce any synergism. The combinations did not cause any changes in DNA condensing ability measured with ethidium bromide exclusions. The proton pump inhibitor, bafilomycin A1, had similar effects in both cell lines. Interestingly, the combination of Dosper (a positive control) and PEI caused the most effective transfection synergism in the presence of serum, although Lipofectamine, with or without PEIs, was a very potent reagent demonstrating the best transfection efficiency in the absence of serum. It is suggested that the PEI/Dosper-mediated synergism in the transfection efficiency may be a general mechanism for liposomal transfection reagents, although the effects can vary depending on cell lines.

Topics: Animals; beta-Galactosidase; DNA; Drug Carriers; Drug Synergism; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Lipids; Liposomes; Macrolides; Muscle, Smooth; Polyethyleneimine; Quaternary Ammonium Compounds; Rabbits; Rats; Transfection; Tumor Cells, Cultured

A retroviral vector constructed from the murine leukemia virus (MLV) can only express transgenes in cells undergoing mitosis, indicating its suitability as a delivery vehicle for cancer gene therapy. However, the transduction efficiency (TE) of retroviruses embedding endogenous envelope proteins in human cancer cells was found to be unsatisfactory. Recently, several research groups have demonstrated the feasibility of a retroviral vector pseudotyped with a vesicular stomatitis virus G (VSV-G) protein. In this study, the potential of VSV-G pseudotyped MLV-based retrovirus was examined as a delivery vehicle in a variety of human cancer cells including brain tumor cells in vitro and in vivo. The transduction efficiency of the 293T/G/GP/LacZ retrovirus in cell culture was superior in most cancer cells, particularly in brain tumor cells, compared with that of other retroviruses, such as PA317- or PG13-derived. The relative growth rate and phosphatidylserine expression level on the plasma membrane of target cells mainly influenced the transduction efficiency of VSV-G pseudotyped retrovirus, which suggested that both the relative growth rate and phosphatidylserine expression level were major determinants of TE. Furthermore, 293T/G/GP/LacZ could efficiently transduce human cancer cells regardless of the presence of chemical additives, whereas in other retroviruses, cationic chemical additives such as polybrene or liposomes were essential during virus infection. Finally, an average of 10% gene expression was routinely obtained exclusively in the tumor mass when 293T/G/GP/LacZ concentrated by simple ultracentrifugation was directly administrated to pre-established brain tumors in animal models (U251-N nu/nu mice or C6 Wistar rats). All told, the present study suggests that the VSV-G pseudotyped retrovirus is a suitable vector for brain tumor gene therapy.

Topics: Brain Neoplasms; Cation Exchange Resins; Cell Membrane; Cholesterol; Fatty Acids, Monounsaturated; Genetic Therapy; Genetic Vectors; Hexadimethrine Bromide; Humans; Leukemia Virus, Murine; Lipids; Phosphatidylserines; Protamines; Quaternary Ammonium Compounds; Transduction, Genetic; Tumor Cells, Cultured; Vesicular stomatitis Indiana virus

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-beta-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 microl) and DNA (3 and 6 microg). Transfection efficiency was quantified by microscopic evaluation of beta-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express beta-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 microl DNA and 18 microl DOTAP/ml (1:6 ratio), 6 microg DNA and 12 microl DOSPER/ml (1:2 ratio), or 6 microg DNA and 12 microl Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8+/-0.6 (mean +/- SEM) for DOTAP-transfected islets compared with 8.4+/-0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1+/-0.5 and 3.4+/-0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

Topics: Animals; beta-Galactosidase; Cation Exchange Resins; Dogs; Fatty Acids, Monounsaturated; Gene Expression Regulation; Genetic Vectors; Glucose; Insulin; Insulin Secretion; Islets of Langerhans; Lipids; Liposomes; Quaternary Ammonium Compounds; Transfection; Viruses

Interleukin (IL)-10 is an anti-inflammatory cytokine that has great potential for use in the treatment of inflammatory and immune illnesses. In this study, gene transfer was used to induce IL-10 transgene expression in murine lungs for treatment of endotoxin-induced lung inflammation. Gene transfer was performed with a cytomegalovirus (CMV)-IL-10 plasmid with the aid of the liposomal agents LipofectAMINE and N-[1-(2,3-dioleoyl)propyl]-N,N, N-trimethylammonium methylsulfate (DOTAP). Administration of the endotoxin caused a marked increase in lung inflammation as indicated by increased tumor necrosis factor (TNF)-alpha release and neutrophil count. Pretreatment of the mice with IL-10 plasmid with and without LipofectAMINE had no inhibitory effect on lung inflammation and IL-10 transgene expression. LipofectAMINE by itself induced lung inflammation, an effect that was not observed with DOTAP. IL-10 plasmid when codelivered with DOTAP expressed biologically active IL-10 protein and caused a reduction in endotoxin-induced inflammation. Transgene expression was observed as early as 3 h after administration, peaked at 12 h, and declined thereafter. We conclude that IL-10 gene transfer is a feasible approach for the treatment of lung inflammation.

Topics: Animals; Cation Exchange Resins; Cytomegalovirus; Endotoxins; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Inflammation; Interleukin-10; Lipids; Lipopolysaccharides; Liposomes; Lung; Male; Mice; Mice, Inbred BALB C; Plasmids; Quaternary Ammonium Compounds

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in

Topics: Anti-Bacterial Agents; Cation Exchange Resins; Cell Line; Cell Nucleus; Chloroquine; Cytochalasin B; Cytoplasm; DNA; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Fatty Acids, Monounsaturated; Gene Expression Regulation; Humans; Kidney; Lipids; Liposomes; Luciferases; Macrolides; Mitosis; Nocodazole; Promoter Regions, Genetic; Protamines; Quaternary Ammonium Compounds; Subcellular Fractions

Topics: Animals; Cation Exchange Resins; Cells, Cultured; Dogs; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Vectors; Islets of Langerhans; Islets of Langerhans Transplantation; Lipids; Quaternary Ammonium Compounds

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Topics: Animals; Cation Exchange Resins; COS Cells; DNA; Drug Carriers; Drug Stability; Fatty Acids, Monounsaturated; Freezing; Lipids; Liposomes; Quaternary Ammonium Compounds; Transfection

We have examined lipids as transfection agents to introduce recombinant plasmids into primary cultures of rat hippocampal neurons. By modifying the protocol for transfection mediated by the commercial reagent DOTAP, we were able to achieve a transfection efficiency of about 3%. Expression of various transfected gene products was sustained for several weeks in culture, the neurons developed normally and the transfected gene products were targeted to the appropriate subcellular compartment.

Topics: Animals; Cation Exchange Resins; Cells, Cultured; DNA; Drug Carriers; Fatty Acids, Monounsaturated; Fluorescent Antibody Technique; Fluorescent Dyes; Glycine; Hippocampus; Immunoglobulin kappa-Chains; Lipids; Mice; Neurons; Plasmids; Quaternary Ammonium Compounds; Rats; Recombinant Proteins; Spermine; Transfection

Direct in vivo transfection of tumor nodules in situ via liposome-DNA complexes has been employed as a strategy to accomplish antitumor immunization. To circumvent the potential safety hazards associated with systemic localization of delivered DNA, the utility of mRNA transcript-mediated gene delivery was explored. Capped, polyadenylated mRNA transcripts encoding the firefly luciferase and Escherichia coli lacZ reporter genes were derived by in vitro transcription. Transfection of the human breast cancer cell line MDA-MB-435 in vitro was accomplished employing cationic liposome-mRNA complexes. Evaluation of a panel of cationic liposome preparations demonstrated significant differences in the capacity of the various preparations to accomplish mRNA-mediated transfection. Quantitative evaluation of in vitro transfection demonstrated that target cells could be transfected at a high level of efficiency. The mRNA liposome-complexes were evaluated for in vivo transfection of tumor nodules in human xenografts in athymic nude mice. It could be demonstrated the liposome-mRNA complexes were comparable in efficacy to liposome-DNA complexes in accomplishing in situ tumor transfection. Thus, mRNA may be considered as an alternative to plasmid DNA as a gene transfer vector for genetic immunopotentiation applications.

Topics: beta-Galactosidase; Breast Neoplasms; Cation Exchange Resins; Cations; Drug Carriers; Fatty Acids, Monounsaturated; Genes, Reporter; Genetic Therapy; Glycine; Humans; Lipids; Liposomes; Luciferases; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; RNA, Messenger; Safety; Spermine; Transfection; Tumor Cells, Cultured