Pilot Project #4

Year 2013-2014

Hypoxia-sensitive long circulating multifunctional nanopreparations for siRNA delivery

Project Investigator: Dr.Can Sarisozen

         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.


1. Synthesis of PEG-Az-PEI-DOPE / PEG-PEI-DOPE / rhodamine-labeled PEI and rhodamine labeled polymers
2. Preparation of polyplexes, ethidium bromide assay and RNase protection.
  • 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
3. Physicochemical characterization of micellar nanopreparations
  • Size and zeta distribution by DLS, morphology investigation by AFM
4. Confirmation of hypoxic conditions by detection of pimonidazole adducts
5. Interactions between cells and micellar nanopreparations under normoxic and hypoxic conditions using FACS and CLSM
  • Fluorescently-labeled formulations (including micellar polyplexes prepared with FAM-labeled siRNA), different incubation times, N/P ratios
6. Survivin silencing experiments
  • 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
3. Detection of apoptosis after survivin downregulation
  • Annexin V/propidium iodide staining and Caspase-3 activity
7. Cytotoxicity of survivin siRNA containing formulations
8. Preparation of antibody modified stimuli-sensitive formulations
  • AntiCA-IX or antiGLUT-1 antibody modified formulations will be prepared using Mal-PEG-azobenzene-PEI-DOPE polymer and Traut’s reagent activated antibody.

All experiments will be done using MDR (and their sensitive counterparts as controls) cell monolayers and spheroids under hypoxic and normoxic conditions.



  • Contact:

    Marina Sheynina
    Phone: 617-373-6004
    Fax: 617-373-7509