Search

Future improvements of non-viral vectors for siRNA delivery require better understanding of intracellular processing and vector interactions with target cells. Here, we have compared the siRNA delivery properties of a lipid derivative of bPEI 1.8kDa (DOPE-PEI) with branched polyethyleneimine (bPEI) with average molecular weights of 1.8kDa (bPEI 1.8kDa) and 25kDa (bPEI 25kDa). We find mechanistic differences between the DOPE-PEI conjugate and bPEI regarding siRNA condensation and intracellular processing. bPEI 1.8kDa and bPEI 25kDa have similar properties with respect to condensation capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8kDa improves the siRNA delivery properties, but with markedly different formulation requirements and mechanisms of action compared to conventional PEIs. Interestingly, strong knockdown using bPEI 25kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH., Statement of Significance: Gene silencing therapy has the potential to treat diseases which are beyond the reach of current small molecule-based medicines. However, delivery of the small interfering RNAs (siRNAs) remains a bottleneck to clinical implementation, and the development of safe and efficient delivery systems would be one of the most important achievements in medicine today. A major reason for the lack of progress is insufficient understanding of cell-polyplex interaction. We investigate siRNA delivery using polyethyleneimine (PEI) based vectors and examine how crucial formulation parameters determine the challenges associated with PEI as a delivery vector. We further evaluate how lipid conjugation of PEI influences formulation, cytotoxicity and polymer interaction with cells and cargo as well as the proton sponge capabilities of the vectors., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The clinical application of gene silencing mediated by small interfering RNA (siRNA) has been limited by the lack of efficient and safe carriers. Phospholipid modification of low molecular weight polyethylenimine (PEI 1.8 kDa) dramatically increased its gene down-regulation capacity while keeping cytotoxicity levels low. The silencing efficacy was highly dependent on the nature of the lipid grafted to PEI and the polymer/siRNA ratio employed. Phosphoethanolamine (DOPE and DPPE) and phosphocholine (PC) conjugation did not change the physicochemical properties and siRNA binding capacity of PEI complexes but had a large impact on their transfection and ability to down-regulate Green Fluorescent Protein (GFP) expression (60%, 30% and 5% decrease of GFP expression respectively). We found that the micelle-forming structure of DOPE and DPPE-PEI dramatically changed PEI's interaction with cell membranes and played a key role in promoting PEI 1.8 kDa transfection, completely ineffective in the absence of the lipid modification., From the Clinical Editor: While siRNA-based gene silencing methods could have numerous clinical applications, efficient delivery remains a major challenge. This team reports that DOPE-PEI and DPPE-PEI based micelle-forming nanostructures may be able to provide an efficient vector for siRNA transfection., (© 2014.)