The main goal of UNIT 1 is to develop a liposome-based platform for active and selective delivery of condensed siRNA particles to cancer stem cells in vivo. This platform should further be capable of (1) efficient encapsulation and triggered release of bioactive siRNA complexes, and (2) enhanced biostability in vivo.
NanoCAN's UNIT 1 is located at the MEMPHYS - Center for Biomembrane Physics, Department of Physics and Chemistry, University of Southern Denmark.
The use of liposomes as delivery vehicles for siRNA-condensates is a very promising approach furnished by the favorable properties of the liposomes, like being biocompatible and biodegradable as well as providing a wide range of properties. The liposomal platform has as a main goal to provide tools for improving the siRNA's biostability, pharmacokinetics, targeting, and intracellular delivery.
In terms of size and properties, the liposomes will be adapted to their final purpose. Regarding tumor stages with sufficient size and vascularization, e.g. the primary tumor, the liposomes are expected to passively accumulate in the vicinity of cancer cells due to the leaky vascular and impaired lymphatic drainage in cancer tissues, the so-called enhanced permeability and retention (EPR) effect. THE EPR effect causes that particles of a certain size range conveniently enter the tumor based on increased vascular permeability and are hindered to exit the tumor based on impaired lymphatic drainage in cancer tissues. The net effect is an enrichment in the cancer tissue. For targeting cancer stem cell in metastases, which may not be sufficiently vascularized to make use of the EPR effect, alternative strategies are to be developed.
The in vivo stability and pharmacokinetics of the siRNA-carrying nano-lipo-complexes can be improved via the incorporation of a hydrophilic polymer, like polyethylene glycol (PEG) or hyaluronic acid (HA). Active targeting can be accomplished by covalent attachment of a targeting ligands, like antibodies and aptamers, which are specific to cell surface markers, to the surface of liposomes. An efficient encapsulation of siRNA will be accomplished by condensation of siRNA using polycations like poly-ethyleneimine (PEI) or protamine. Last and not least, standardized model systems are set up to test and compare the performance of the various permutations of liposomal systems in vitro and in vivo. The standardized cell models are developed in UNIT 4 and in vivo testing is performed in UNIT 6.
Fig. 1: (Move mouse into image to magnify). Schematic representation of the fabrication of a liposomal based drug delivery platform utilizing siRNA. Large unilamellar vesicles [LUVs] are formed from the extrusion of multilamellar vesicles [MLVs]. Addition of either pegylated phospholipids or hyaluronic acid will increase the circulation time in the cardiovascular system. Specificity is achieved by covalently attaching either aptamers or antibodies to the surface of the liposome. Finally, the liposomes can be loaded with, for instance, PEI:siRNA polymer complexes by rehydrating lyophilized liposomes in the presence of pre-condensed PEI:siRNA polyplexes.
Fig.2: A cartoon showing the principle of the luciferase assay. The assay uses luciferase (Luc2 gene) transfected MCF7 cells. When luciferin is added to the cells, it will be enzymatically oxidized by luciferase in an ATP-driven process and light will be released. The knockdown of the luc2 gene will reduce, and may as well terminate, the production of the luciferase enzyme in the cells, which will eventually affect the observed light signal.