title: Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.
abstract: The feasibility of exploiting secretory phospholipase A2 (sPLA2) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.
authors.affiliation: MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark; Lundbeckfonden Center of Excellence NanoCAN, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Arouri
authors.affiliation: Lundbeckfonden Center of Excellence NanoCAN, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark; Molecular Oncology Group, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors.forename: Jakub
authors.initials: J
authors.lastname: Trojnar
authors.affiliation: Lundbeckfonden Center of Excellence NanoCAN, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark; Molecular Oncology Group, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark; Lundbeckfonden Center of Excellence NanoCAN, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors.forename: Anders H
authors.initials: AH
authors.lastname: Hansen
authors.affiliation: Lundbeckfonden Center of Excellence NanoCAN, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark; Molecular Oncology Group, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Mollenhauer
authors.affiliation: MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark; Lundbeckfonden Center of Excellence NanoCAN, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.
authors.forename: Ole G
authors.initials: OG
authors.lastname: Mouritsen
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002626:Chemistry, Pharmaceutical; D002784:Cholesterol / Q000737:chemistry; D004337:Drug Carriers / Q000378:metabolism*; D065546:Drug Liberation / Q000502:physiology*; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D008163:Luminescent Measurements; D061986:MCF-7 Cells; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D054467:Phospholipases A2 / Q000032:analysis*
chemical_list: D004337:Drug Carriers; D008081:Liposomes; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C084352:distearoyl phosphatidylglycerol; D002784:Cholesterol; C010942:1,2-distearoyllecithin; D054467:Phospholipases A2