Design of polymer brush vectors for controlled RNA delivery: insights from PDMAEMA and PDIPA systems in physiologically relevant conditions Carlos Eduardo Neri-Cruz , Julien E. Gautrot School of Engineering and Materials Science, Queen Mary University of London, UK Towards the design of hierarchical macromolecular architectures block copolymers for enhanced and sustained RNA delivery, we studied the kinetics for the binding of siRNA and mRNA to single-blocks of poly(2- (dimethylamino ethyl)methacrylate) (PDMAEMA) and poly(2-(diisopropylamino)ethyl methacrylate) (PDIPA) brushesunder neutral (pH 7.4) and acidic (pH 5.5) conditions in PBS, using a validated SPR-based method 1 . For this study, polymer brushes of 30 nm were synthesised through SI-ATRP and characterised beforehand via ellipsometry, XPS, and FTIR. In accordance with the solution conformation assessed by ellipsometry, more swollen PDMAEMA chains at neutral pH displayed superior capacity for complexing RNA molecules, in contrast to hydrophobic PDIPA chains. An increased absorption of RNA to PDIPA was observed in acidic conditions, as a result of the transition to the hydrophilic state 2 . Interestingly,in acidic conditions, binding capacity of siRNA to PDIPA equalled the one observed from PDMAEMA at neutrality. Protonation of PDMAEMA and PDIPA brushes also led to improved complexation of mRNA, however, this was still limited by the inherent steric hindrance associated to large RNA molecules 3 , and apparently, intensifiedby the bulky isopropyl groups of PDIPA. Comparison between siRNA and mRNA binding at neutral pH revealed that the absorption of the latter with PDIPA was faster and more stable (lower desorption), contrary to PDMAEMA complexes. This finding was unexpected, considering the large size of mRNA, and might be related to different chemical affinities exhibited from single and double strands of RNA to PDIPA. Since the current assays confirmed the low affinity of RNA to PDIPA, at physiological pH, our future research will focus on studying PDMAEMA-b-PDIPA systems, where PDMAEMA serves as the capture component, while PDIPA aims to reduce the competitive binding at the cytosol, particularly, by small RNA competitors triggering the destabilisation of the brush-RNA complex 4 .We will additionally explore the potential sustained RNA release and shielding ability of larger PDIPA brushes, when collapsed in a di-block system. Even though opening of PDIPA chains at the endosome environment might result on eventualdeshielding, highly protonated chains might simultaneously, accelerate the endosomal escape by enhanced electrostatic-mediated membrane disruption 5 . References 1. F. Qu, D. Li, X. Ma, F. Chen and J. E. Gautrot, Biomacromolecules , 2019, 20 , 2218-2229. 2. J. D. Willott, B. A. Humphreys, T. J. Murdoch, S. Edmondson, G. B. Webber and E. J. Wanless, Phys Chem Chem Phys , 2015, 17 , 3880-3890. 3. M. Krishnamoorthy, D. Li, A. S. Sharili, T. Gulin-Sarfraz, J. M. Rosenholm and J. E. Gautrot, Biomacromolecules , 2017, 18 , 4121-4132. 4. A. A. M. Raynold, D. Li, L. Chang and J. E. Gautrot, Nat Commun , 2021, 12 , 6445. 5. I. M. S. Degors, C. Wang, Z. U. Rehman and I. S. Zuhorn, Acc Chem Res , 2019, 52 , 1750-1760.
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