11624 DOI: 10.1021/la101806z Langmuir 2010, 26(14), 11624–11627 Published on Web 06/15/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society PEGylated Amyloid Peptide Nanocontainer Delivery and Release System V. Castelletto, J. E. McKendrick, and I. W. Hamley* Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, U.K. U. Olsson and C. Cenker Physical Chemistry 1, Lund University, S-221 00 Lund, Sweden Received May 6, 2010. Revised Manuscript Received June 7, 2010 A micellar nanocontainer delivery and release system is designed on the basis of a peptide-polymer conjugate. The hybrid molecules self-assemble into micelles comprising a modified amyloid peptide core surrounded by a PEG corona. The modified amyloid peptide previously studied in our group forms helical ribbons based on a β-sheet motif and contains β-amino acids that are excluded from the β-sheet structure, thus being potentially useful as fibrillization inhibitors. In the model peptide-PEG hybrid system studied, enzymatic degradation using R-chymotrypsin leads to selective cleavage close to the PEG-peptide linkage, break up of the micelles, and release of peptides in unassociated form. The release of monomeric peptide is useful because aggregation of the released peptide into β-sheet amyloid fibrils is not observed. This concept has considerable potential in the targeted delivery of peptides for therapeutic applications. Introduction The conjugation of polymers to peptides or proteins enables the creation of novel nanomaterials for applications in drug delivery, 1 cell growth media, 2-5 tissue scaffolding, 6,7 and others. This sub- ject has been reviewed. 8-12 Conjugates containing poly(ethylene glycol) (PEG) are of particular interest because of the biocompat- ibility, availability, and well-known physicochemical properties of this polymer. 13-17 Here, we first show that the conjugation of PEG to peptide βAβAKLVFF leads to the formation of spherical micelles. This was unexpected, given our recent work that shows that when PEG3000 is conjugated to peptides such as FFKLVFF a fibril structure is retained. 18,19 Core-shell fibrils self-assemble with a peptide core and a PEG corona. We then demonstrate that the enzymatic cleavage of βAβAKLVFF-PEG using enzyme R-chy- motrypsin (RC) leads to the breakup of the spherical micelles producing fragments βAβAKLVF and F-PEG3000. R-Chymo- trypsin selectively cleaves peptide bonds following aromatic residues 20 and is therefore expected to cleave the F-F bond preferentially. (It may also cleave after the L residue.) Peptide βAβAKLVFF is under study in our group 21 because it comprises a self-recognition motif, KLVFF, that can bind to the correspond- ing sequence Aβ(16-20) in the amyloid β(Aβ) peptide conjugated to two β 2 -alanine residues that cannot participate in the β-sheet hydrogen-bonding pattern. Furthermore, β-amino acid peptides are resistant to proteolysis, so the N-terminus of this model compound contains residues that will promote bioavailability. A recent report describes the use of an enzyme to cleave a peptide-polymer conjugate into its constituent peptide and polymer fragments. A L 4 K 8 L 4 peptide was linked to PEG3000 via a linker containing a substrate (RG unit) for thrombin enzymatic cleavage. 22 A transition from R-helical to β-sheet secondary structure following enzyme treatment was indicated by circular dichroism (CD), and AFM showed fibrils, consistent with the self-assembly of the β-sheets into amyloid fibrils. Enzy- matic dephosphorylation has been reported to act as a switch to enable the self-assembly of a phosphothreonine-containing pep- tide in a PEG-peptide conjugate, driving self-assembly into β-sheet fibrils. 23 Enzyme-responsive vesicular polymeric nano- containers are well known. 24 However, we are not aware of previous reports on peptide-PEG micellar nanocontainers for enzyme-controlled peptide release. In the present work, we show that enzyme treatment can release a peptide from the core of the micellar nanocontainers after the detachment of PEG chains. Our system is designed according to *Corresponding author. E-mail: i.w.hamley@reading.ac.uk. Also at Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0DE, U.K. (1) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347–360. (2) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Science 2009, 234, 59–63. (3) Mata, A.; Hsu, L.; Capito, R.; Aparicio, C.; Henrikson, K.; Stupp, S. I. Soft Matter 2009, 5, 1228–1236. (4) Spoerke, E. D.; Anthony, S. G.; Stupp, S. I. Adv. Mater. 2009, 21, 425–430. (5) Todd, S. J.; Scurr, D. J.; Gough, J. E.; Alexander, M. R.; Ulijn, R. V. Langmuir 2009, 25, 7533–7539. (6) Place, E. S.; Evans, N. D.; Stevens, M. M. Nat. Mater. 2009, 8, 457–470. (7) Place, E. S.; George, J. H.; Williams, C. K.; Stevens, M. M. Chem. Soc. Rev. 2009, 38, 1139–1151. (8) L€ owik, D. W. P. M.; Ayres, L.; Smeenk, J. M.; van Hest, J. C. M. Adv. Polym. Sci. 2006, 202, 19–52. (9) Klok, H.-A.; Schlaad, H. Peptide Hybrid Polymers; Springer: Berlin, 2006; Vol. 202. (10) Van Hest, J. C. M. J. Macromol. Sci., Part C: Polym. Rev. 2007, 47, 63–92. (11) Gauthier, M. A.; Klok, H. A. Chem. Commun. 2008, 23, 2591–2611. (12) Lutz, J. F.; B€ orner, H. G. Prog. Polym. Sci. 2008, 33,1–39. (13) Zalipsky, S. Adv. Drug Delivery Rev. 1995, 16, 157–182. (14) Veronese, F. M. Biomaterials 2001, 22, 405–417. (15) Greenwald, R. B. J. Controlled Release 2001, 74, 159–171. (16) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discovery 2003, 2, 214–221. (17) Torchilin, V. P. Pharm. Res. 2007, 24,1–16. (18) Hamley, I. W.; Krysmann, M. J.; Castelletto, V.; Noirez, L. Adv. Mater. 2008, 20, 4394–4397. (19) Hamley, I. W.; Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Noirez, L.; Hule, R. A.; Pochan, D. Chem. ; Eur. J. 2008, 14, 11369–11374. (20) Creighton, T. E. Proteins: Structures and Molecular Properties; W. H. Freeman: New York, 1993. (21) Castelletto, V.; Hamley, I. W.; Hule, R. A.; Pochan, D. J. Angew. Chem., Int. Ed. 2009, 48, 2317–2320. (22) Koga, T.; Kitamura, K.-I.; Higashi, N. Chem. Commun. 2006, 4897–4899. (23) K€ uhnle, H.; B€ orner, H. G. Angew. Chem., Int. Ed. 2009, 48, 6431–6434. (24) Nardin, C.; Thoeni, S.; Widmer, G.; Winterhalter, M.; Meier, W. Chem. Commun. 2000, 1433–1434.