Hypoxia-sensitive long circulating multifunctional nanopreparations for siRNA delivery
One of the projects within the Center of Translational Nanotechnology Excellence (NHI funded CCNE at Northeastern University) is focused on developing PEG-PE based polymeric micelles for the protection and delivery of siRNA. Here, wepropose long-circulating stimuli-sensitive (hypoxia sensitive) nanopreparations based on self-assembling PEG-azobenzene-PEI-lipid conjugates, which can complex siRNA via its positive charge. When such preparations accumulate in tumors via the enhanced permeability and retention (EPR) effect, in hypoxic tumor areas PEG groups would be detached from PEI-lipid/siRNA complexes because of azobenzene linker degradation, leading to the exposure of PEI’s positive charge to promote cellular internalization of remaining complexes and their release into the cell cytoplasm because of PEI’s proton sponge effect. In preliminary experiments we found that PEI-lipid conjugates could form micelles and successfully contained, protected and delivered siRNA. Also, synthesis of hypoxia-sensitive PEG-azobenzene PEI-lipid conjugate could successfully achieved. Under hypoxic conditions, degradation of the PEG groups was investigated and self-assembled hypoxia-sensitive micelle structures were able to deliver GFP siRNA into the cells under hypoxic conditions. We hypothesize that these systems can be successfully used to deliver therapeutic siRNAs into the cells via their stimuli-sensitive properties and their potential can be further reinforced by the addition of targeting moieties such as anti-CA IX or anti-GLUT-1 antibodies. Assessment of the formulations will be performed using a broad set of MDR cancer cells in vitro under hypoxic conditions using normoxia and drug sensitive cancer cells as controls. Moreover, 3D multi cellular cancer spheroids as intermediate models of hypoxic tumors will also be used to evaluate the formulations potential.
- Complexation of siRNA will be assessed by EtBr exclusion
- Reversibility of complex formation will be demonstrated by fluorescence recovery after heparin treatment
- Protection against RNAse will be evaluated by incubation of free and complexed siRNA with an RNAse cocktail followed by electrophoresis on agarose gel containing EtBr with non-damaged siRNA as control
- Size and zeta distribution by DLS, morphology investigation by AFM
- Fluorescently-labeled formulations (including micellar polyplexes prepared with FAM-labeled siRNA), different incubation times, N/P ratios
- Survivin silencing effect of the hypoxia-sensitive formulations will be analyzed by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), western blot and ELISA
- Annexin V/propidium iodide staining and Caspase-3 activity
- AntiCA-IX or antiGLUT-1 antibody modified formulations will be prepared using Mal-PEG-azobenzene-PEI-DOPE polymer and Traut’s reagent activated antibody.