1-2-dioleoyloxy-3-(trimethylammonium)propane and Uterine-Cervical-Neoplasms

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I-restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8(+) splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8(+) tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8(+) TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8(+) splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8(+) TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8(+) anti-tumor response.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; CD8 Antigens; Cytokines; Dendritic Cells; Fatty Acids, Monounsaturated; Female; Interferon-alpha; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Papillomavirus E7 Proteins; Quaternary Ammonium Compounds; T-Lymphocytes, Cytotoxic; Uterine Cervical Neoplasms

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