Production and characterization of sulforhodamine B containing large unilamellar vesicles labeled with atrazine Original Research Article

As an essential part in the development of an immunological based dipstick test for screening the presence of hormone disrupting pseudo-estrogens in aqueous samples, liposomes containing sulforhodamine B and labeled with atrazine as a model compound were produced. The hapten–lipid conjugate (atrazine-dipalmitoyl phosphatidyl ethano-lamine, DPPE) is obtained by coupling atrazine to DPPE with a C6 spacer in between. Both the atrazine-C6 spacer intermediate as well as the final reaction product are characterized by TLC, HPLC, GC-MS and 1H-NMR, respectively. The production of the liposomes is based on the reverse-phase evaporation technique. Each individual step of the production is optimized, e.g. the manner of addition of the phases, the duration of sonication and evaporation, the type of recipient, the duration of vortex-mixing and final evaporation, the number of consecutive filtrations, the composition of the dialysis buffer, the ultracentrifugation speed as well as the type of antioxidant used. The obtained liposomes are characterized by electron microscopy and by dynamic light scattering (diameter and size-distribution). In addition, we also determined the lipids, phospholipids, cholesterol, the percentage of oxidized lipids, the amount of encapsulated dye and the quantity of liposomes produced. Ten separate productions spread over different days and following the described optimized procedure yielded a reproducible production of large unilamellar vesicles (diameter: 654 ± 109 nm). Additional characteristics of one single production include: 3.9 × 1011 liposomes/ml; 2.4 × 106 molecules of dye inside; a calculated amount of 1.85 × 104 molecules of atrazine outside.