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

Application of superparamagnetic iron oxide nanoparticles (SPIOs) as the contrast agent has improved the quality of magnetic resonance (MR) imaging. Low efficiency of loading the commercially available iron oxide nanoparticles into cells and the cytotoxicity of previously formulated complexes limit their usage as the image probe. Here, we formulated new cationic lipid nanoparticles containing SPIOs feasible for in vivo imaging.. Hydrophobic SPIOs were incorporated into cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and polyethylene-glycol-2000-1,2-distearyl-3-sn-phosphatidylethanolamine (PEG-DSPE) based micelles by self-assembly procedure to form lipid-coated SPIOs (L-SPIOs). Trace amount of Rhodamine-dioleoyl-phosphatidylethanolamine (Rhodamine-DOPE) was added as a fluorescent indicator. Particle size and zeta potential of L-SPIOs were determined by Dynamic Light Scattering (DLS) and Laser Doppler Velocimetry (LDV), respectively. HeLa, PC-3 and Neuro-2a cells were tested for loading efficiency and cytotoxicity of L-SPIOs using fluorescent microscopy, Prussian blue staining and flow cytometry. L-SPIO-loaded CT-26 cells were tested for in vivo MR imaging.. The novel formulation generates L-SPIOs particle with the average size of 46 nm. We showed efficient cellular uptake of these L-SPIOs with cationic surface charge into HeLa, PC-3 and Neuro-2a cells. The L-SPIO-loaded cells exhibited similar growth potential as compared to unloaded cells, and could be sorted by a magnet stand over ten-day duration. Furthermore, when SPIO-loaded CT-26 tumor cells were injected into Balb/c mice, the growth status of these tumor cells could be monitored using optical and MR images.. We have developed a novel cationic lipid-based nanoparticle of SPIOs with high loading efficiency, low cytotoxicity and long-term imaging signals. The results suggested these newly formulated non-toxic lipid-coated magnetic nanoparticles as a versatile image probe for cell tracking.

Topics: Adenocarcinoma; Animals; Cations; Cell Differentiation; Cell Line, Tumor; Colorectal Neoplasms; Contrast Media; Fatty Acids, Monounsaturated; Feasibility Studies; Ferric Compounds; HeLa Cells; Humans; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Imaging; Male; Metal Nanoparticles; Mice; Mice, Inbred BALB C; Micelles; Neuroblastoma; Particle Size; Phosphatidylethanolamines; Polyethylene Glycols; Prostatic Neoplasms; Quaternary Ammonium Compounds; Rhodamines

Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo.. DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed.. The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group.. The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.

Topics: Adenocarcinoma; Apoptosis; Cell Division; Cholesterol; Fatty Acids, Monounsaturated; Female; Genes, p53; Genetic Vectors; Humans; Liposomes; Ovarian Neoplasms; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Various strategies have been attempted to design efficient protocols for ovarian cancer gene therapy but there has been little progress in their clinical application. In this study, we formulated and evaluated a new cationic liposome prepared with dioleoyltrimethylaminopropane (DOTAP), 1,2-dioleoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol) (DDC) for plasmid DNA transfer into ovarian cancer cells.. The DDC liposome was prepared by mixing the DOTAP:DOPE:Cholin a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pEGFP-C1) and DDC were complexed at various weight ratios to find the optimum condition and the percentage of transfected cells was determined by selecting a green fluorescence protein (GFP) expressing cells in flow cytometry. The transfection efficiency of the DDC liposome was compared with 3[N-(N,N-dimethylaminoethylene) carbamoyl] cholesterol (DC-Chol)/DOPE liposome and commercially available lifopectin.. The optimal transfection of plasmid DNA was achieved at a 1:4 (w/w) ratio of DDC to DNA. The DDC/DNA complex exhibited higher transfection efficiency in human ovarian cancer cells (OVCAR-3 and SK-OV-3 cells) compared to that in other types of cell lines (NCI-NIH:522 and HepG2 cells). Flow cytometric analysis revealed that the DDC/DNA complex exhibited an over fourfold increase in GFP expression levels compared with DC-Chol/DOPE or lipofectin in OVCAR-3 cells. This result was further confirmed by confocal microscopy and RT-PCR analysis.. These results suggest that our newly formulated cationic liposome (DDC) appears to be a promising nonviral vector for treating ovarian adenocarcinoma because of its selective high gene transfer ability in ovarian cancer cells.

Topics: Adenocarcinoma; Cations; Cholesterol; DNA; Fatty Acids, Monounsaturated; Female; Genetic Therapy; Genetic Vectors; Glycerophospholipids; Green Fluorescent Proteins; Humans; Lipids; Liposomes; Luminescent Proteins; Ovarian Neoplasms; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured

We have prepared a panel of lipidic ammonium tetrafluoroborate salts that contain trifluoromethyl, trichloromethyl, and methyl groups attached to the headgroup. 19F-NMR analyses of the cationic lipid panel revealed that the differences in electron-withdrawal from the ammonium ion headgroup accounted for differences in ion-pairing. Exchange of the tetrafluoroborate counterion by complexation to DNA-phosphate of a reporter gene enabled us to probe the influence of inductive electron-withdrawal in cationic lipid-mediated DNA transfection. We tested the lipid panel for transfection activity in two cell lines. The results indicate that the inductive effects of electron-withdrawing functionality diminish transfection activity in modest (2-4-fold) increments. The present study suggests that the mechanism whereby poly(alcohol)- or poly(ether)-substituted headgroups improve DNA transfection is not based on electronic activation of the ammonium ion.

Topics: Adenocarcinoma; Borates; Boric Acids; Breast Neoplasms; Cations; DNA; Fatty Acids, Monounsaturated; Fluorescent Dyes; Humans; Lipids; Magnetic Resonance Spectroscopy; Phosphates; Quaternary Ammonium Compounds; Transfection; Tumor Cells, Cultured

The power of antisense phosphodiester oligonucleotides (aODN) as regulatory molecules of gene expression is strongly limited by their low cellular uptake and very rapid nuclease-mediated degradation. This study deals with the effect of artificial cationic lipids on ODN cellular uptake and degradation in cell cultures and in human serum. At the ODN levels normally used in antisense-mediated gene regulation experiments, a cationic lipid, DOTAP, enhances the rate of ODN uptake more than 25 fold, but at lower ODN levels the effect of DOTAP is absent. These findings are consistent with a mechanism of ODN internalization by receptor-mediated saturable endocytosis that is bypassed by DOTAP. ODN degradation by nucleases is markedly prevented by DOTAP both in cultured cells and in human serum. Other cationic lipids, namely DOTMA and DOGS, exhibit very similar behaviour. The relatively slight cellular toxicity revealed by cationic lipids contribute to render these molecules very suitable for aODN vehiculation.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Base Sequence; Biological Transport; Cell Line; Colonic Neoplasms; Drug Resistance; Fatty Acids, Monounsaturated; Glycine; Humans; Kinetics; Leukemia; Mice; Molecular Sequence Data; Oligonucleotides, Antisense; Quaternary Ammonium Compounds; Spermine; T-Lymphocytes; Time Factors; Tumor Cells, Cultured