Inhibition of Amyloid  Protein Fibrillation by Polymeric Nanoparticles Celia Cabaleiro-Lago, †,§ Fiona Quinlan-Pluck, †,§ Iseult Lynch, † Stina Lindman, ‡ Aedin M. Minogue, § Eva Thulin, ‡ Dominic M. Walsh, § Kenneth A. Dawson,* ,† and Sara Linse ‡ Centre for BioNano Interactions, School of Chemistry and Chemical Biology, UniVersity College Dublin, Belfield, Dublin 4, Ireland, Department of Biophysical Chemistry, Lund UniVersity, P.O. Box 124, 22100 Lund, Sweden, and Laboratory for NeurodegeneratiVe Research, UCD Conway Institute for Biomolecular and Biomedical Research, UniVersity College Dublin, Belfield, Dublin 4, Ireland Received June 3, 2008; E-mail: kenneth@fiachra.ucd.ie Abstract: Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer’s disease-associated amyloid  protein (A). We found that these nanoparticles affect mainly the nucleation step of A fibrillation. The elongation step is largely unaffected by the particles, and once the A is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of A is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that A binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric A and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of A initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear. Introduction The ability of proteins to form amyloid fibrils seems to be largely sequence-independent, and many proteins can form structures with characteristic cross- stacking perpendicular to the long axis of the fiber. 1-3 While the molecular events behind the processes leading from native to fibrillar states remain elusive, data accumulated from many studies suggest that fibrillation involves a number of intermediate oligomeric states of different association numbers and structures. 4,5 The use of agents that interfere with these processes and/or allow for the isolation of intermediate species may help elucidate the mo- lecular mechanism of fibril formation. Such strategies also have therapeutic potential for the treatment of amyloidosis. We recently identified copolymeric nanoparticles as agents that accelerate the fibrillation of 2microglobulin (2m). 6 Specifically, we found that the presence of nanoparticles leads to a shortening of the lag phase for nucleation of the fibrillation process. The likely role of the nanoparticles in this process is binding of 2m, which increases the local concentration and the likelihood of formation of a critical nucleus for fibrillation. 6 Depending on the relative affinity of nanoparticles for protein monomers, unfolded monomers, oligomers, critical nuclei, and other prefibrillar states, the influence of nanoparticles on protein fibrillation kinetics is likely to be protein-dependent. Quite different effects may also be anticipated if the protein species are bound in a similar conformation as in solution or if the binding process alters the conformation of the protein or causes local or global unfolding. Thus, to further our understanding of protein fibrillation processes and how these are perturbed by the presence of nanoparticles, it is necessary to collect large amounts of well-controlled data in a protein- and particle- dependent manner. In the present study, we have investigated the effect of copolymeric N-isopropylacrylamide: N-tert-butylacrylamide (NiPAM:BAM) nanoparticles having a nominal size of 40 nm on the fibrillation of amyloid  protein (A). Previous studies in the literature regarding A and nanoparticles have focused on detection and diagnosis 7,8 or targeting and destruction 9,10 of amyloid aggregates, and only a few papers have investigated † School of Chemistry and Chemical Biology, University College Dublin. ‡ Department of Biophysical Chemistry, Lund University. § UCD Conway Institute for Biomolecular and Biomedical Research, University College Dublin. (1) Kelly, J. W. Structure 1997, 5, 595–600. (2) Sunde, M.; Blake, C. AdV. Protein Chem. 1997, 50, 123–159. (3) Teplow, D. B. Amyloid 1998, 5, 121–142. (4) Chiti, F.; Dobson, C. M. Annu. ReV. Biochem. 2006, 75, 333–366. (5) Stefani, M.; Dobson, C. M. J. Mol. Med. 2003, 81, 678–699. (6) Linse, S.; Cabaleiro-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S. E.; Dawson, K. A Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8691–8696. (7) Choi, J. S.; Choi, H. J.; Jung, D. C.; Lee, J. H.; Cheon, J. Chem. Commun. 2008, 2197–2199. (8) Haes, A. J.; Hall, W. P.; Chang, L.; Klein, W. L.; Van Duyne, R. P. Nano Lett. 2004, 4, 1029–1034. (9) Bastus, N. G.; Kogan, M. J.; Amigo, R.; Grillo-Bosch, D.; Araya, E.; Turiel, A.; Labarta, A.; Giralt, E.; Puntes, V. F. Mater. Sci. Eng., C 2007, 27, 1236–1240. (10) Olmedo, I.; Araya, E.; Sanz, F.; Medina, E.; Arbiol, J.; Toledo, P.; Alvarez-Lueje, A.; Giralt, E.; Kogan, M. J. Bioconjugate Chem. 2008, 19, 1154–1163. Published on Web 10/28/2008 10.1021/ja8041806 CCC: $40.75  2008 American Chemical Society J. AM. CHEM. SOC. 2008, 130, 15437–15443 9 15437