1-2-dioleoyloxy-3-(trimethylammonium)propane and Liver-Neoplasms

Hepatocellular carcinoma (HCC), a common deadly malignancy, requires novel therapeutic strategies to improve the survival rate. Combining chemotherapy and gene therapy is a promising approach for increasing efficiency and reducing side effects. We report on the design of highly specific lipid nanoparticles (LNPs) encapsulating both the chemotherapeutic drug, sorafenib (SOR), and siRNA against the midkine gene (MK), thereby conferring a novel highly efficient anticancer effect on HCC. The LNPs were modified with a targeting peptide, SP94, which is selective for hepatic cancer cells (HCCs), thus permitting the specific delivery of the payload. MK-siRNA increased the sensitivity of HCCs, HepG2, to SOR (IC

Topics: Carcinoma, Hepatocellular; Cell Survival; Combined Modality Therapy; Drug Delivery Systems; Drug Liberation; Drug Synergism; Fatty Acids, Monounsaturated; Gene Knockdown Techniques; Gene Silencing; Genetic Therapy; HeLa Cells; Hep G2 Cells; Humans; Inhibitory Concentration 50; Lipids; Liver Neoplasms; Midkine; Nanoparticles; Piperidines; Quaternary Ammonium Compounds; RNA, Small Interfering; Sorafenib

To enhance the solubility of rosiglitazone, rosiglitazone-loaded cationic lipid emulsion was formulated using cationic lipid DOTAP, DOPE, castor oil, tween 20, and tween 80. The formulation parameters in terms of droplet size were optimized focused on the effect of the cationic lipid emulsion composition ratio on drug encapsulating efficiency, in vitro drug release, and cellular uptake of the rosiglitazone-loaded emulsion. Droplet sizes of a blank cationic emulsion and a rosiglitazone-loaded cationic emulsion ranged between 195-230 nm and 210-290 nm, respectively. The encapsulation efficiency of the rosiglitazone-loaded emulsion was more than 90%. The rosiglitazone-loaded cationic emulsion improved in vitro drug release over the drug alone and showed a much higher cellular uptake than rosiglitazone alone. Moreover, drug loading in cationic emulsions increased cellular uptake of rosiglitazone in insulin-resistant HepG2 cells more than the normal HepG2 cells. Taken together, these results indicate that cationic lipid emulsions could be a potential delivery system for rosiglitazone and could enhance its cellular uptake efficiency into target cells.

Topics: Biological Transport; Carcinoma, Hepatocellular; Castor Oil; Cations; Chemistry, Pharmaceutical; Drug Carriers; Drug Stability; Emulsions; Fatty Acids, Monounsaturated; Hep G2 Cells; Humans; Hydrogen-Ion Concentration; Hypoglycemic Agents; Insulin Resistance; Lipids; Liver Neoplasms; Nanoparticles; Particle Size; Phosphatidylethanolamines; Polysorbates; Quaternary Ammonium Compounds; Rosiglitazone; Solubility; Surface-Active Agents; Technology, Pharmaceutical; Thiazolidinediones

The glycyrrhetinic acid-modified stealth cationic liposomes (GA-PEG-CLs) loaded with pDNA (GA-PEG-CLPs) were developed and found to transfect human hepatocellular carcinoma cell line HepG2 with high efficiency. GA-PEG-CLs were comprised of DOTAP, cholesterol (Chol) and glycyrrhetinic acid-polyethyleneglycol-cholesterol conjugate (GA-PEG-Chol). Agarose gel electrophoresis revealed that 5% GA-PEG-CLs constituted by DOTAP/Chol/GA-PEG-Chol at molar ratio of 50:45:5 could completely entrap pDNA at a lower liposomes/pDNA weight ratios of 4:1 (N/P ratio: 1.14). Compared to ordinary cationic liposomes (CLs), steric cationic liposomes (PEG-CLs) and 1% GA-PEG-CLs made from DOTAP/Chol/MPEG2000-Chol/GA-PEG-Chol at molar ratio of 50:45:4:1, 5% GA-PEG-CLs were found to possess the highest transfection efficiency as gene vectors in serum-free or serum-containing medium in PKCalpha over-expressed HepG2 cells but no significance difference in human embryonic kidney cell line HEK 293. Additionally, 5% GA-PEG-CLs have the lowest cytotoxicity on human normal hepatocyte cell line L02. The competitive inhibition experiments mediated by GA were carried out in HepG2 cells, which demonstrated that GA-PEG-CLs could deliver selectively pDNA to hepatoma cells by the targeting moiety GA. In conclusion, GA-PEG-CLs containing 5% GA-PEG-Chol might be one of the most potential gene vectors as hepatoma targeting therapy.

Topics: Carcinoma, Hepatocellular; Cations; Cholesterol; Fatty Acids, Monounsaturated; Genetic Therapy; Genetic Vectors; Glycyrrhetinic Acid; HEK293 Cells; Hep G2 Cells; Humans; Liposomes; Liver Neoplasms; Particle Size; Polyethylene Glycols; Quaternary Ammonium Compounds; Transfection

Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary.. Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process.. We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-gamma were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth.. In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity.

Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Carcinoma, Hepatocellular; Fatty Acids, Monounsaturated; Female; Fetuins; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Interleukin-12; Lactic Acid; Liver Neoplasms; Mice; Mice, Inbred BALB C; Nanoparticles; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Quaternary Ammonium Compounds; Transgenes; Tumor Cells, Cultured

In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25kDa) or linear (22kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.

Topics: Cations; Cell Line, Tumor; Cell Survival; Cholesterol; Deoxyribonuclease I; DNA; Electrochemistry; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Humans; Interleukin-12; Lipids; Liver Neoplasms; Particle Size; Polyethyleneimine; Polymers; Quaternary Ammonium Compounds; Serum; Transfection

Recently, cationic steroids have been developed that display broad-spectrum antibacterial activity. These compounds, characterized by the presence of several amino groups, present a facially amphiphilic morphology. Formulations containing such steroids were tested for their ability to facilitate the uptake of a reporter plasmid into various cell lines. The results show that, when associated with the naturally occurring zwitterionic lipid dioleoyl-phosphatidylethanolamine (DOPE), cationic steroid antibiotics allow for transfection levels comparable to those obtained with DOTAP. The activity of the amphiphilic mixture was nearly unaffected by bafilomycin A1 and chloroquine treatment, suggesting a mechanism that is independent of the acidification process associated with endocytosis. Collectively, our results show that DNA delivery agents possessing strong antibacterial properties can be obtained by conjugating amino groups to a steroid nucleus.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Carcinoma, Hepatocellular; Cell Line; Cell Line, Transformed; Cell Line, Tumor; DNA; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genes, Reporter; Humans; Liver Neoplasms; Luciferases; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Steroids; Transfection

The receptor-ligand interaction between hepatocyte heme receptors and heme was evaluated as a basis for developing a targeted cationic lipid delivery reagent for nucleic acids. Heme (ferric protoporphyrin IX) was conjugated to the aminolipid dioleoyl phosphatidylethanolamine (DOPE) and used to form cationic lipid particles with dioleoyl trimethylammonium propane (DOTAP). These lipids particles (DDH) protect oligoribonucleotides from degradation in human serum and increase oligoribonucleotide uptake into 2.2.15 human hepatoma cells (to a level of 50-60 ng oligo/10(4) cells) when compared with the same lipid particles (DD) prepared identically without heme. The DDH heme level that was optimal for oligoribonucleotide delivery was also optimal for maximum expression of plasmid-encoded luciferase. The enhancing effect of heme was evident only at net particle negative charge. Fluorescence microscopy showed that DDH delivered oligoribonucleotides into both the 2.2.15 cell cytoplasm and nucleus. DDH may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in such liver diseases as viral hepatitis, hepatoma, and hypercholesterolemia.

Topics: Animals; Carcinoma, Hepatocellular; Cations; Cell Line; Cell Nucleus; Chlorocebus aethiops; Cytoplasm; DNA, Recombinant; Drug Carriers; Fatty Acids, Monounsaturated; Genes, Reporter; Genetic Vectors; Heme; Humans; Kidney; Liver Neoplasms; Luciferases; Mice; Microscopy, Fluorescence; Neoplasm Proteins; Oligoribonucleotides; Organ Specificity; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Receptors, Cell Surface; Recombinant Fusion Proteins; Species Specificity; Tumor Cells, Cultured; Vero Cells

Plasmid DNA condensed by polylysine enhanced cationic-liposome-mediated transfection of Hep G2 cells. The luciferase expression plasmid pCMVL was complexed with the polycation poly-L-lysine and mixed with liposomes that contained a 1:1 molar ratio of the cationic lipid 1,2-dioleoyloxy-3-trimethyl-ammoniumpropane, with the neutral phospholipid 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phosphoethanolamine. Polylysine enhanced cationic-liposome-mediated transfection of the hepatoblastoma cell line Hep G2 9-fold compared with pCMVL complexed alone with liposomes. The ratio of cationic to anionic charge of the polylysine-pCMVL complexes, and the quantity of cationic liposomes, are important determinants for optimal transfection of Hep G2 cells.

Topics: Cations; Electrophoresis, Agar Gel; Fatty Acids, Monounsaturated; Hepatoblastoma; Humans; Liposomes; Liver Neoplasms; Luciferases; Particle Size; Phosphatidylethanolamines; Plasmids; Polylysine; Quaternary Ammonium Compounds; Transfection; Tumor Cells, Cultured