Diethanolamine Chloride Research Articles

Therapeutic effects and anti-metastasis effects of cationic liposomes against pancreatic cancer metastasis in vitro and in vivo

The anti-metastatic effects of cationic liposomes (CL) composed of 87 mol% dimyristoylphosphatidylcholine (DMPC), 8 mol% O,O′-ditetradecanoyl-N-(α–trimethylammonioacetyl) diethanolamine chloride (2C14ECl) and 5 mol% polyoxyethylene(21) dodecyl ether (C12(EO)21) was investigated for human pancreatic cancer (BxPC-3) cells. The inhibitory effect of CL on the migration of BxPC-3 cells was observed based on a wound scratch assay. CL suppressed pseudopodium formation of BxPC-3 cells. The anti-invasive effect of CL against BxPC-3 cells was observed via a Matrigel invasion assay. The anti-invasive effect of CL for BxPC-3 cells was found to occur through the inhibition of MMP2, MMP9, and MMP14. Overall, the results of this study revealed for the first time, the therapeutic effects and anti-metastasis activity of CL in xenograft mouse models for peritoneal metastasis of human pancreatic cancer.

Negatively Charged Cell Membranes-Targeted Highly Selective Chemotherapy with Cationic Hybrid Liposomes against Colorectal Cancer In Vitro and In Vivo

Cationic hybrid liposomes (CHL) composed of 87 mol% L-α-dimyristoylphosphatidylcholine (DMPC), 5 mol% polyoxyethylene (21) dodecyl ether (C12(EO)21) and 8 mol% O,O’-ditetradecanoyl-N-(α–trimethylammonioacetyl) diethanolamine chloride (2C14ECl) were prepared by the method of sonication. A clear solution of CHL having a hydrodynamic diameter of 100 nm could be maintained over 4 weeks. The IC50 value of CHL on the growth of human colorectal cancer (CRC; HCT116) cells was remarkably smaller than that of the DMPC liposomes. Immediate fusion of CHL including NBDPC into HCT116 cell membranes was confirmed using a confocal laser and total internal reflection fluorescence (TIRF) microscope without affecting normal colon (CCD-33Co) cells. Activation of caspases for apoptosis of HCT116 cells induced by CHL was verified on the basis of fluorescence microscopy. Carcinoma HCT116 cells have lower membrane potential compared to normal CCD-33Co cells, since negatively charged PS and GM1 in HCT116 cells increased compared with that of normal CCD-33Co. Therapeutic effects of CHL were obtained in xenograft mouse models of CRC in vivo. Induction of apoptosis in tumor of xenograft mouse model of human CRC treated with CHL was observed in micrographs of tissue section on the basis of TUNEL method. Furthermore, no severe side effects of CHL were observed in toxicity tests using normal mice.

Optimization of siRNA Delivery Method into the Liver by Sequential Injection of Polyglutamic Acid and Cationic Lipoplex

Previously, we developed a novel siRNA transfer method to the liver by sequential intravenous injection of poly-L-glutamic acid (PGA) and cationic liposome/siRNA complex (cationic lipoplex). In this study, we examined the effects of the charge ratio (+/-) of cationic liposome/siRNA, molecular weight of PGA and cationic lipid of cationic liposome on the biodistribution of siRNA after sequential injection of PGA plus cationic lipoplex. When 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (Chol) lipoplex was intravenously injected into mice, the accumulation of siRNA was mainly observed in the lungs. In contrast, when DOTAP/Chol lipoplex was intravenously injected at 1 min after intravenous injection of PGA, siRNA was largely accumulated in the liver. The charge ratio (+/-) of DOTAP/Chol liposome/siRNA did not affect the biodistribution of siRNA after sequential injection. As regards the molecular weight of PGA, the accumulation of siRNA was observed mainly in the liver after the sequential injection of PGA of 20.5, 38, 64 or 200 kDa plus DOTAP/Chol lipoplex. Furthermore, to examine the effect of cationic lipid of cationic liposome on the biodistribution of siRNA, we prepared other cationic liposomes composed of 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride (DOTMA)/Chol, dimethyldioctade-cylammonium bromide (DDAB)/Chol and O,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)di-ethanolamine chloride (DC-6-14)/Chol. For the cationic liposomes, the accumulation of siRNA was observed mainly in the liver when their cationic lipoplexes were sequentially injected after injection of PGA into mice. From these findings, sequential injection of PGA plus cationic lipoplex could deliver siRNA efficiently into the liver regardless of the charge ratio (+/-) of lipoplex, lengths of PGA and cationic lipid of liposome.

Therapeutic Effects of Cationic Hybrid Liposomes on the Hepatic Metastasis of Colon Carcinoma Along with Apoptosis <i>in Vivo</i>

Therapeutic effects of cationic hybrid liposomes (HL) composed of 87 mol% dimyristoyl-phosphatidylcholine (DMPC), 5 mol% polyoxyethylene (21) dodecyl ether (C12(EO)21) and 8 mol% O,O'-ditetradecanoyl-N-(α-trimethyl-ammonioacetyl) diethanolamine chloride (2C14ECl) on the metastasis of human colon carcinoma (HCT116) cells were examined in vivo. Cationic HL having a hydrodynamic diameter less than 150 nm were preserved for one month. Therapeutic effects were obtained in the hepatic metastasis mouse models of HCT116 cells after the intravenous injection of cationic HL. The histological analysis indicated the induction of apoptosis in the liver section of the hepatic metastasis mouse models treated with cationic HL in vivo. Therapeutic effects of cationic HL without any drugs on the hepatic metastasis were revealed for the first time on the basis of histological analyses in vivo.

Antimicrobial activity of the ionic liquids triethanolamine acetate and diethanolamine chloride, and their corresponding Pd(II) complexes

The antimicrobial activity of the ionic liquids triethanolamine acetate [TEA][HOAc] and diethanolamine chloride [HDEA][Cl], as well as of their Pd(II) complexes trans-dichlorobis(triethanolamine-N)palladium(II) (trans-[PdCl2(TEA)2]) and diethanolammonium–tetrachloridopalladate(II) ([HDEA]2[PdCl4]), is presented. The investigated compounds showed low antibacterial activity. Better results were for antifungal activity. [TEA][HOAc] exhibited better activity than corresponding complex. Aspergillus species were especially sensitive to [HDEA]2[PdCl4]. The activity of this complex against A. restrictus, A. fumigatus was up to ten times higher than the activity of positive control, fluconazole.

Anti-tumor Effects of Cationic Hybrid Liposomes against Colon Carcinoma along with Apoptosis <i>in Vitro</i>

New-type three-component cationic hybrid liposomes (HLs) composed of dimyristoylphosphatidylcholine (DMPC), polyoxyethylene(21)dodecyl ether (C(12)(EO)(21)) and O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine chloride (2C(14)ECl) were produced. Cationic HLs were smaller and more stable than pure DMPC liposomes. It is noteworthy that cationic HLs could remarkably inhibit the growth of human colon cancer (HCT116) cells along with apoptosis in vitro for the first time in this study.

Gene transfer effects on various cationic amphiphiles in CHO cells

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O'-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.

Ultrasound enhances liposome-mediated gene transfection

Previous studies have shown that some series of liposomes, usually containing cationic lipids, are useful tools for gene introduction into cells. To investigate the effect of ultrasound (US) on liposome-mediated transfection, three types of liposomes (designated L1, L2 and L3, in the order of increasing transfection efficiency) containing O, O′-ditetradecanoyl- N-(α-trimethylammonioacetyl) diethanolamine chloride, dioleoylphosphatidylethanolamine, and/or cholesterol at varying ratios, were used in this study. HeLa cells were treated with liposome–DNA complexes containing luciferase genes for 2 h before sonication. Optimal US condition for the enhancement was determined to be 0.5 W/cm 2, 1 MHz continuous wave for 1 min and was above threshold for inertial cavitation based on EPR detection of free radicals. Luciferase expressions 24 h after the treatments were significantly increased by sonication to 2.4 fold with L1, and 1.7 fold with L2. However, with L3, which showed the highest level of expression among the liposomes, significant but minimal enhancement was observed when sonication was done 15 min after the DNA-L3 treatment, suggesting that efficiency of the liposome also determines the proper timing for sonication. The 2 h pre-sonication incubation with liposome–DNA complexes for L1 and L2 (30 min for L3) required to attain enhancement, suggests that US works to enhance transfection only after cells had enough DNA uptake.

An assessment of relative transcriptional availability from nonviral vectors

To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O, O′-ditetradecanoyl- N-(α-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression.

Characteristics and biodistribution of cationic liposomes and their DNA complexes

We have developed some novel liposome formulations for gene transfection. The formulations consisting of O, O′-ditetradecanoyl- N-(α-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, phospholipid and cholesterol showed effective gene transfection activity in cultured cells with serum and in vivo, i.e., intraperitoneal injection in mice. In this report, the physicochemical characteristics and biodistribution of the liposomes containing DC-6-14 (DC-6-14 liposomes) as a drug (gene) carrier for gene therapy were investigated in vitro and in vivo. DC-6-14 liposome–DNA complexes were usually thought to have positive surface charge. However, depending on the ratio of DNA to liposomes, zeta-potential of the complexes became negative. The diameter of the complexes also depended on the DNA–liposome ratio, and showed a maximum when their surface potential was neutral. When biodistribution of the complexes was determined after intravenous injection, positively charged complexes showed an immediate lung accumulation. On the other hand, negatively charged complexes did not show lung accumulation. These results have suggested that biodistribution of the DNA–liposome complexes, prepared with DC-6-14 liposomes, depends on their surface charge. Therefore, some surface modification of DC-6-14 liposomes may improve the biodistribution and hence the targetability of their DNA complexes.

A new cationic liposome for efficient gene delivery with serum into cultured human cells: a quantitative analysis using two independent fluorescent probes

Cationic liposomes are useful to transfer genes into eukaryotic cells in vitro and in vivo. However, liposomes with good transfection efficiency are often cytotoxic, and also require serum-free conditions for optimal activity. In this report, we describe a new formulation of cationic liposome containing DC-6–14, O, O′-ditetradecanoyl- N-(α-trimethylammonioacetyl)diethanolamine chloride, dioleoylphosphatidylethanolamine and cholesterol for gene delivery into cultured human cells. This liposome, dispersed in 5% serum-containing growth medium, efficiently delivered a plasmid DNA for GFP (green fluorescent protein) into more than 80% of the cultured human cell hybrids derived from HeLa cells and normal fibroblasts. Flow cytometric analysis revealed that the efficiency of the GFP gene expression was 40–50% in a tumor-suppressed cell hybrid, while it was greatly reduced in the tumorigenic counterpart. The enhanced GFP expression in tumor-suppressed cell hybrids was quantitatively well correlated with a prolonged presence of the plasmid DNA, which had been labeled with another fluorescent probe, ethidium monoazide, within the cells. These results suggest that a newly developed cationic liposome is useful for gene delivery in serum-containing medium into human cells and the stability of the plasmid DNA inside the cell is a crucial step in this liposome-mediated gene expression. The mechanisms by which cationic liposome mediates gene transfer into eukaryotic cells are also discussed.

Development of novel cationic liposomes for efficient gene transfer into peritoneal disseminated tumor.

A novel series of cationic lipids has been found, by in vivo screening, to be effective for gene transfer into peritoneal disseminated tumor. O,O'-Ditetradecanoyl-N-(alpha-trimethylammonioacetyl)diethan olamine chloride (DC-6-14), having dimyristyl acid, has shown the highest transfection activity in vitro, provided that 10% fetal bovine serum is present. To enhance the transfection efficiency of DC-6-14, we added dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (Chol) as helper lipids in various ratios. Cationic liposomes containing DC-6-14, DOPE, and Chol in molar ratios of 1:0.75:0.75 and 1:1:0.8 maintained efficient transfection activity under serum-containing conditions in HRA, mEIIL, and ES-2 cell lines in vitro, as determined by luciferase assay. With our novel liposomes, transfection efficiencies were higher in cells proliferating faster than in cells proliferating slower, depending on mitotic activity as represented by labeling index. In the mEIIL peritoneal disseminated tumor model, cancer cells were specifically transfected with the lacZ gene. Gene transfer was observed by X-Gal staining not only in floating cancer cells in the ascites, but also in the peritoneal disseminated cancer tissue. The percentage of LacZ-positive cells was about 1%, which was significantly higher than with commercially available Lipofectin (0.38%), LipofectACE (0.62%), or LipofectAMINE (0.23%). In the mEIIL peritoneal disseminated tumor-nude mouse model, herpes simplex thymidine kinase gene (HSV tk) transfer with our novel liposomes, followed by ganciclovir (GCV) treatment, resulted in significantly longer survival compared with control mice (p < 0.05, Cox-Mantel). These results suggest that these liposomes show promise as tools in gene therapy for patients with intraperitoneal disseminated cancer.