Synthesis of a library of strictly linear, redox-sensitive poly(ethylene imine)-9-poly(ethylene glycol) block copolymers for enhanced tissue distribution of polyplexes
Final Report Abstract
Due to their ability to alter structures and processes in a biological context, nucleic acids such as plasmid DNA or siRNA have the potential to become a completely new class of drugs. The major challenge towards their clinical application is the fabrication of the efficient and safe delivery systems. On the way to this overall goal, we proposed three specific aims: The first idea involved the development of reductively degradable PEI-based polymers. To this end, low MW, linear PEI building blocks were connected with varying amounts of disulfide containing cross-linkers to receive a straightforward polymer library. The hypothesis was that the degradable polymers are perfectly suited to form polyplexes that are stable in the extracellular environment, but are rapidly disassembled inside cells. These polymers have great promise for the delivery of plasmid DNA. In six out of the seven cell lines the reductively degradable PEIs were highly superior to seven commercially available transfection reagents and at the same time substantially less toxic. Another hypothesis of the reductively cleavable polymers was that the intracellular polymer degradation would allow for a significantly higher release of the nucleic acid compared to non-degradable PEIs. Confocal laser scanning microscopy experiments supported the hypothesis. The microscopic images illustrated the distribution and availability over the whole cell on a sufficiently short time scale, which is a necessary event for the activity of siRNA. Besides these favorable results, both studies greatly contributed to establishing design criteria for nucleic acid transporting carriers: the implementation of redox-sensitive moieties into the carrier is highly promising to achieve three goals in one sweep: a high efficacy, a low toxicity, and a significant intracellular nucleic acid release. This clearly rendered polymer degradation based on disulfides superior to hydrolytically labile groups, which have only a gradual degradation kinetic on a much larger time scale. The second idea involved the shielding of PEI-polyplexes using PEG to prevent unwanted and unspecific interactions with non-target cells. To this end, a library of 39 strictly linear, non-degradable PEG-PEI diblock copolymers was synthesized. In contrast to other approaches, the copolymers demonstrated a clear separation between the PEG and PEI moieties. The hypothesis was that the copolymers are perfectly suited to systematically investigate trends concerning the physicochemical properties and the biological activity of the gene carriers. Very small (< 150 nm) and neutral polyplexes were fabricated using PEG-PEI copolymers with a PEG content higher than 50%. Because the transfection efficacy of these polyplexes was significantly reduced compared to the PEI homopolymer, reductively degradable PEG-PEI copolymer counterparts were synthesized. These copolymers reesthablished the transfection efficacy and suggested that the intracellular removal of the PEG domain is a essential for the transfection process. By the screening of this large copolymer library, valuable design criteria for the optimization of the shielding moiety of gene delivery carriers were gained. The third idea aimed at improving the nanoparticles´ mobility in the ECM. To this end, PEG molecules of various MW were tethered to positively charged model nanoparticles at varying densities from 0.4 – 30 PEG/nm2. The extraordinary high PEG density is most likely due to the unique surface texture of the model nanoparticles and still under investigation. For testing the nanoparticles mobility, a straightforward set-up using cells embedded in Matrigel in combination with FRAP measurements was applied. A PEGylation of positively charged silica nanoparticles using PEG with a MW of 5 kDa at a PEG density of 12 PEG/nm2 or higher was identified as optimal for sufficient nanoparticle mobility in the ECM. Despite such a high PEG density on the surface, the nanoparticles were still taken up by cells in a 3D context. This was the first time that the influence of nanoparticle PEGylation on their mobility in the ECM was investigated in such a systematic manner. Our approach can easily be applied for screening the mobility of other nanoparticle species. In conclusion, with our studies we unveiled basic principles for the design of efficient and safe materials for plasmid DNA and siRNA delivery.
Publications
- Bioabbaubare Polymere als Genfähren für die nichtvirale Transfektion. Pharmazeutische Zeitung, 153(5): 288-295 (2007)
M. Breunig, U. Lungwitz, A. Goepferich
- Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci USA, 104(36): 14454-14459 (2007)
M. Breunig, U. Lungwitz, R. Liebl, A. Goepferich
- Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: disulfide bonds boost intracellular release of the cargo. J Control Release, 130(1): 57-63 (2008)
M. Breunig, C. Hozsa, U. Lungwitz, K. Watanabe, I. Umeda, H. Kato, A. Goepferich
- Delivery of nucleic acids via disulfidebased carrier systems. Adv Mater, 21(32-33): 3286-3306 (2009)
S. Bauhuber, C. Hozsa, M. Breunig, A. Goepferich
- Enhancing the intracellular release of siRNA with biodegradable poly(ethylene imine) as a carrier system, J Control Release Newsletter, 26(2): 4-5 (2009)
M. Breunig, C. Hosza, U. Lungwitz, K. Watanabe, I. Umeda, H. Kato, A. Goepferich
- A library of strictly linear poly(ethylene glycol)-poly(ethylene imine) diblock copolymers to perform structure function relationship of non-viral gene carriers. J Control Release 162(2): 446-455 (2012)
S. Bauhuber, R. Liebl, L. Tomasetti, R. Rachel, A. Goepferich, M. Breunig
(See online at https://doi.org/10.1016/j.jconrel.2012.07.017)
