1-2-dielaidoylphosphatidylethanolamine and Breast-Neoplasms

The aim of this study is to develop targeted nanoliposome formulations to provide efficient treatment for breast cancer. In this study, peptide 18-modified poly(2-ethyl-2-oxazoline)-dioleoylphosphatidylethanolamine (P18-PEtOx-DOPE), was synthesised to construct nanoliposomes.. Doxorubicin (DOX) was encapsulated into the nanoliposomes by ethanol injection method. Particle size and polydispersity index were measured by dynamic light scattering. Zeta potential was determined by electrophoretic laser Doppler anemometry. The shape of the nanoliposomes was examined by transmission electron microscope. Specific bindings of P18-PEtOx-DOPE nanoliposomes were demonstrated on AU565 cells by confocal microscopy and flow cytometry studies.. DOX-loaded nanoliposomes with particle diameter of 150.00 ± 2.84 nm and PDI of 0.212 ± 0.013 were obtained. PEtOx-DOPE and PEtOx-DOPE nanoliposomes are non-toxic on HUVEC, HEK293 and hMSC cells for 48 h. Furthermore, P18-PEtOx-DOPE nanoliposomes demonstrated specificity towards AU565 cells with high binding affinity.. As a result, DOX-loaded P18-PEtOx-DOPE nanoliposomes can serve as favourable candidates in breast cancer targeted therapy.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Delivery Systems; Drug Design; Drug Screening Assays, Antitumor; Drug Stability; Female; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Liposomes; Myocytes, Smooth Muscle; Nanoparticles; Oxazoles; Particle Size; Peptides; Phosphatidylethanolamines; Scattering, Radiation

In the present study, we constructed a tumor-targeting and pH-sensitive lipoprotein-mimic nanocarrier containing paclitaxel (FA-BSA-LC/DOPE-PTX), by adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and oleic acid as pH-sensitive components into the formulation of lipid core and then coating with folic acid modified bovine serum albumin (FA-BSA) for tumor targeting activity. In vitro drug release study demonstrated that paclitaxel (PTX) was released from FA-BSA-LC/DOPE in a pH-dependent manner. The vitro cytotoxicity assays showed that all the blank nanocarriers were nontoxic. However, MTT assay showed that FA-BSA-LC/DOPE-PTX was highly cytotoxic. Cellular uptake experiments analyzed with flow cytometry and laser scan confocal microscope (LSCM) revealed that FA-BSA-LC/DOPE was taken up in great amount via folate receptor-mediated endocytosis and pH-sensitive release of drug to cytoplasm. Furthermore, the study of intracellular drug release behavior demonstrated that the FA-BSA-LC/DOPE escaped from lysosomes and released drug into cytoplasm. The in vivo targeting activity showed that the nanocarrier selectively targeted tumor and had long residence time for BSA layer increased the stability in blood. Moreover, FA-BSA-LC/DOPE-PTX produced very marked anti-tumor activity in tumor-bearing mice in vivo. Therefore, FA-BSA-LC/DOPE as biocompatible, tumor-targeting and pH-sensitive lipoprotein-mimic nanocarrier is a promising system for effective intracellular delivery of PTX to tumor.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cytoplasm; Drug Carriers; Drug Delivery Systems; Female; Folic Acid; Humans; Hydrogen-Ion Concentration; Lipids; Lysosomes; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Oleic Acid; Paclitaxel; Phosphatidylethanolamines; Serum Albumin, Bovine

The cationic lipid mediated uptake of plasmid DNA by cells in monolayer culture was significantly enhanced with an aqueous solution of the block copolymer poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol) (PPDO/PLLA-b-PEG). Plasmid uptake studies with DNA encoding the beta-galactosidase gene and cytotoxicity evaluations were performed on MCF-7, NIH 3T3 and CT-26 cell lines. Transfection yields and time courses for maximum release of FITC labeled DNA in MCF-7 cells were observed and quantified by beta-galactosidase assay and spectrofluorometry, respectively. The reported results suggest that the studied block copolymer might be useful for the enhancement of polycation mediated transfection and could find application in gene therapy.

Topics: 3T3 Cells; Animals; Biological Transport; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; DNA; Humans; Kinetics; Materials Testing; Mice; Phosphatidylethanolamines; Plasmids; Polyesters; Polyethylene Glycols; Transfection

Viral vector systems are the most commonly used gene transfer tools for clinical gene therapy. However, lipofection systems are potential alternatives because of lower immunogenicity and easier cGMP production, but in vivo stability and transduction efficacy need to be improved. Therefore, we investigated gene transduction efficiency of our novel cGMP cationic lipids, CCQ22 and CCQ32, by FACS analysis. Toxicity analysis was performed to determine the cytotoxic side effects of the novel lipids. To evaluate the stability of the compounds in the context of local delivery to patients with intraperitoneally metastatic ovarian cancer, gene transfer was also tested in the presence of malignant ascites. Our novel cGMP standard lipids mediated gene transfer rates of more than 50%. However, for most cell lines cytotoxic side effects were similar to our reference lipofection system. In general, ascites had no major influence on gene transduction rates with the novel lipids. Our results suggest that CCQs may compare favorably with commercially available lipofection systems. These promising results facilitate further analysis of the compounds.

Topics: Ascites; Breast Neoplasms; Cell Line, Tumor; Cholesterol Esters; Female; Genetic Vectors; Humans; Lipids; Liposomes; Ovarian Neoplasms; Phosphatidylethanolamines; Plasmids; Transduction, Genetic

Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV)2-luciferase vector using the new nonliposomal transfection reagent FuGene. Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p'-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881. Compared to the traditional calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription. To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast cancer cells with an estrogen response element-luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disruptors for (anti)androgenic and (anti)estrogenic properties.

Topics: Androgen Antagonists; Animals; Breast Neoplasms; CHO Cells; Cricetinae; Drug Evaluation, Preclinical; Environmental Pollutants; Estrogen Antagonists; Genes, Reporter; Humans; Indicators and Reagents; Luciferases; Mammary Tumor Virus, Mouse; Mice; Phosphatidylethanolamines; Receptors, Androgen; Reproducibility of Results; Sensitivity and Specificity; Transfection; Tumor Cells, Cultured

A 28-base phosphodiester triple helix-forming oligonucleotide, mostly G and A containing, targeted to a polypurine tract interrupted by a purine-pyrimidine inversion, situated upstream from the TATA box of the promoter of the human HER2 gene, was conceived by computer modeling. The "energetically best choice" was oligo 28(C), which formed the triple helix in vitro, as proved by gel retardation and Fourier transform infrared spectroscopy. When administered as a complex with lipofectin, fluorescence confocal microscopy and electrophoresis confirmed the delivery and persistence of this unprotected oligonucleotide inside MCF7 (breast cancer) cells. At a concentration of 2 microM, the oligonucleotide reduced within 6 h the HER2 mRNA level to 42% (Northern blot) but did not interfere with the transcription of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. During the first day of administration at 0.22 microM, it lowered to 59% the HER2 protein in treated, as compared to nontreated, cells (ELISA). The effect was sequence specific when compared to that of five different negative controls, and it was target selective when compared to the expression of a related, nontargeted protein, the epidermal growth factor receptor. By day 2, the inhibitory effect was overcome by replenishment reactions.

Topics: Base Sequence; Blotting, Northern; Breast Neoplasms; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides, Antisense; Phosphatidylethanolamines; Purines; Receptor, ErbB-2; Sensitivity and Specificity; Spectroscopy, Fourier Transform Infrared

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