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

The biodistribution of intravenously injected DNA lipid nanocapsules (DNA LNCs), encapsulating pHSV-tk, was analysed by in vivo imaging on an orthotopic melanoma mouse model and by a subsequent treatment with ganciclovir (GCV), using the gene-directed enzyme prodrug therapy (GDEPT) approach. Luminescent melanoma cells, implanted subcutaneously in the right flank of the mice, allowed us to follow tumour growth and tumour localisation with in vivo bioluminescence imaging (BLI). In parallel, DNA LNCs or PEG DNA LNCs (DNA LNCs recovered with PEG(2000)) encapsulating a fluorescent probe, DiD, allowed us to follow their biodistribution with in vivo biofluorescence imaging (BFI). The BF-images confirmed a prolonged circulation-time for PEG DNA LNCs as was previously observed on an ectotopic model of glioma; comparison with BL-images evidenced the colocalisation of PEG DNA LNCs and melanoma cells. After these promising results, treatment with PEG DNA LNCs and GCV on a few animals was performed and the treatment efficacy measured by BLI. The first results showed tumour growth reduction tendency and, once optimised, this therapy strategy could become a new option for melanoma treatment.

Topics: Animals; Benzothiazoles; Carbocyanines; DNA; Electrophoresis, Agar Gel; Fatty Acids, Monounsaturated; Female; Fluorescent Dyes; Ganciclovir; Gene Transfer Techniques; Genes, Transgenic, Suicide; Glycerol; Herpes Simplex; Humans; Lipids; Luciferases; Melanoma-Specific Antigens; Melanoma, Experimental; Mice; Mice, Nude; Molecular Imaging; Nanocapsules; Octoxynol; Oleic Acids; Particle Size; Phosphatidylethanolamines; Plasmids; Polyethylene Glycols; Quaternary Ammonium Compounds; Static Electricity; Stearic Acids; Surface Properties; Thymidine Kinase; Tissue Distribution; Treatment Outcome; Triglycerides; Xenograft Model Antitumor Assays

Fluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1-5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA-) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid-DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies.

Topics: Carbocyanines; DNA; Fatty Acids, Monounsaturated; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Lipids; Liposomes; Osmolar Concentration; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Sodium Chloride; Transfection