O. H. Grandas1, R. K. Fisher1, R. Dieter1, S. S. Kirkpatrick1, J. D. Arnold1, R. M. Buckley1, M. M. McNally1, M. B. Freeman1, D. J. Mountain1 1University Of Tennessee Graduate School Of Medicine,Department Of Surgery,Knoxville, TN, USA
Introduction: RNA interference (RNAi), or the use of short interfering RNA (siRNA) to transiently attenuate cellular protein expression, shows promise as a gene therapy technology to alter vascular pathology. However, the development of molecular nanocarriers is needed to overcome the unfavorable physiochemical properties that limit intracellular siRNA delivery. We have previously established cell penetrating peptide-modified neutral PEGylated liposome (CPP-PLP) as viable molecular nanocarriers in vascular smooth muscle cells. We aim to translate this molecular drug delivery platform for in vivo application in vascular injury models of disease, using liposomes stable when stored for off-the-shelf administration. Here we aim to determine the optimal storage parameters for CPP-PLP stability and siRNA capsulate retention.
Methods: CPP-PLPs were formed as previously described, injected with 200nM siRNA, and stored at room temp, 4°C, or -20°C for 0-48hr in phosphate buffered saline (PBS) or Dulbecco’s Modified Eagle Medium (DMEM). Additionally, 0.1% bovine serum albumin (BSA) in PBS was tested as a stabilizer protein and 0.1% dimethyl sulfoxide (DMSO) was tested in DMEM as a cryoprotectant. Dynamic light scattering (DLS) was used to measure nanoparticle size, size distribution, and nanoparticle fragmentation and/or aggregation. RiboGreen RNA assay was used to determine siRNA encapsulation and retention.
Results: DMSO is ineffective as a cryoprotectant and disrupts liposome membranes leading to siRNA leakage and lysis after 24h storage at all temps. DLS data for DMEM and BSA suggested that morphology was maintained for up to 48hr, but siRNA retention was not improved at any temp. CPP-PLPs stored in PBS exhibited the best siRNA retention after 24 and 48hr storage at 4°C, while storage at -20°C resulted in more siRNA leakage.
Conclusion: Our data suggests the optimal parameters for short-term CPP-PLP/siRNA complex storage are in PBS at 4°C while DMSO, DMEM, and BSA were ineffective at maintaining liposome stability. Further experiments are ongoing to determine siRNA retention under these conditions over time, and how lipid modifications can increase stability for more efficient long-term storage. These studies will direct our experimental implementation of this novel drug delivery technology in future in vivo applications in vascular injury models of disease.