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 subject has been reviewed.8-12 Conjugates containing poly(ethylene glycol) (PEG) are of particular interest because of the biocompatibility, 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 *Corresponding author. E-mail:
[email protected]. 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.
11624 DOI: 10.1021/la101806z
enzymatic cleavage of βAβAKLVFF-PEG using enzyme R-chymotrypsin (RC) leads to the breakup of the spherical micelles producing fragments βAβAKLVF and F-PEG3000. R-Chymotrypsin selectively cleaves peptide bonds following aromatic residues20 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 group21 because it comprises a self-recognition motif, KLVFF, that can bind to the corresponding sequence Aβ(16-20) in the amyloid β(Aβ) peptide conjugated to two β2-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 L4K8L4 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. Enzymatic dephosphorylation has been reported to act as a switch to enable the self-assembly of a phosphothreonine-containing peptide in a PEG-peptide conjugate, driving self-assembly into β-sheet fibrils.23 Enzyme-responsive vesicular polymeric nanocontainers 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 (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.
Published on Web 06/15/2010
Langmuir 2010, 26(14), 11624–11627
Castelletto et al.
Letter
Figure 1. Cryo-TEM images of (a) a 4.2 wt % aqueous solution of βAβAKLVFF-PEG (the inset shows an enlarged image) and (b) a 0.84 wt % aqueous solution of βAβAKLVF. Scheme 1. Concept of an Enzymatic Responsive Peptide Release System Based on a PEGylated Peptide Nanocontainer Delivery Systema
The peptides within the β-strands (red lines) in the core of the micelle (left hand side) are released in unassociated form (right-hand side). a
the concept shown in Scheme 1. PEG provides a “stealth” coating suitable to deliver peptide contained in the cores of micelles, and PEGylation can facilitate enhanced circulation times in vivo.17 The cleaved peptide is released in unassociated form (i.e., not in the form of amyloid fibrils); this is potentially useful in the development of peptide therapeutics. The inability of peptide βAβAKLVF to form β-sheet fibrils, in contrast to βAβAKLVFF, is intrinsically of interest in understanding amyloid self-assembly, and the design principles to achieve this are discussed.
Results and Discussion Mass spectrometry confirms that RC cleaves βAβAKLVFFPEG preferentially at the F-F bond to produce βAβAKLVF and F-PEG3000. Figure S1 shows an ES-MS spectrum with a sharp peak for the peptide at the expected mass and mass distributions as expected for PEG3000 with different m/z values. MS/MS fragmentation spectra in Figures S2 and S3 confirm that the peptide produced contains F, then V, then L at the C terminus. The spectrum is identical to that obtained for a βAβAKLVF peptide synthesized separately. The most direct evidence for a morphology change (on conjugating βAβAKLVFF to PEG and upon enzymatic degradation of the conjugate) is provided by cryo-TEM. Cryo-TEM reveals micelles with a diameter of 8 to 9 nm (Figure 1). Images of βAβAKLVFF were also recorded (separately, see Figure S4 for Langmuir 2010, 26(14), 11624–11627
an example) and compare well to those previously published by us on another batch of the same peptide.21 Clearly, the attachment of PEG drives self-assembly into spherical micelle structures. We propose that PEG forms a shell around a core containing peptides. The dimensions of the micelles are consistent with a core comprising βAβAKLVFF in an extended β-strand and a corona of PEG slightly swollen compared to its radius of gyration. Upon enzymatic cleavage, cryo-TEM reveals the absence of any selfassembled structure on the accessible 1-100 nm length scale (images not shown). Also, no evidence is obtained for self-assembled fibrils of βAβAKLVF (Figure 1b). This is supported by conventional negative stain TEM, which also revealed no visible structures (Figure S5). As a control experiment to check whether PEG can inhibit the fibrillization of the peptide, cryo-TEM was also performed for a mixture of PEG3500 with βAβAKLVF and no structures were observed (Figure S6). Small-angle X-ray scattering (SAXS) is a powerful tool for probing peptide protein structure in solution, avoiding the requirement to crystallize the sample as in X-ray crystallography. SAXS is also well suited to study the structure of PEG/peptide conjugates (drying causes the crystallization of PEG and this disrupts the self-assembled structure in solution25) and the interaction of peptides with enzymes. Here, the technique was used to investigate structural changes upon enzymatic cleavage of βAβAKLVFF-PEG with RC. Figure 2 summarizes the SAXS data. The data show a drastic change in the ordering of selfassembled structures within the solution upon enzymatic cleavage. The data for βAβAKLVFF-PEG before enzymatic cleavage can be fitted to a model form factor used for block copolymer micelles comprising a uniform core to which flexible chains are tethered.26 The dimensions obtained (Table TS1) are in good agreement with those from cryo-TEM. The micelle core radius is 2.5 nm, which corresponds closely to the extended length of a seven-residue peptide.20 The apparent radius of gyration Rg of the attached chains is 5.65 nm, which is larger than value estimated (i.e., Rg PEG(3100) = 2.47 nm), but the effective value allows for swelling in water and packing constraints of the chains at the micelle corecorona interface. Although the solution used for the SAXS experiment is more dilute than the one used for the cryo-TEM experiment, βAβAKLVFF-PEG is above its critical micelle concentration (25) Hamley, I. W.; Krysmann, M. J. Langmuir 2008, 24, 8210–8214. (26) Pedersen, J. S.; Gerstenberg, M. C. Macromolecules 1996, 29, 1363–1365.
DOI: 10.1021/la101806z
11625
Letter
Castelletto et al.
Figure 3. FTIR spectra (top four) for βAβAKLVFF-PEG at Figure 2. SAXS intensity profiles for a 1 wt % solution of βAβAKLVFF-PEG before (0) and after cleavage with RC (red O), along with reference SAXS profiles for a 1 wt % solution of RC alone (blue 4) and the 1 wt % βAβAKLVFF (green O) and 0.9 wt % βAβAKLVF (purple 0) peptide solutions. The solid lines are fits using model form factors described in the text: (red -) form factor fit for βAβAKLVFF and (;) form factor fit for βAβAKLVFF-PEG.
(cmc) as determined by pyrene fluorescence experiments, from which a cmc of 0.28 wt % was determined (Figure S7). The SAXS intensity profile for the enzyme RC in Figure 2 closely resembles the profile measured previously using smallangle neutron scattering (SANS),27 in particular when allowance is made for differences in contrast. (The SANS experiments were carried out in D2O.) Considering peptide βAβAKLVFF, modeling of the SAXS data (excluding points at very low q) produces a very good fit based on a form factor of a cylindrical shell. This is an approximation, because Figure S4 and our previous work shows that βAβAKLVFF forms twisted ribbons that may comprise smaller cylindrical subunits. The inner and outer radii are found to be 4.7 and 5.8 nm, respectively. Other parameters are provided in Table TS1 in the Supporting Information. The thickness of the ribbons (1.1 nm) is approximately equal to the thickness of a β-sheet.28 The twisted ribbon structures visible in Figure S4 may comprise assemblies of smaller subunits; certainly the dimensions are larger than the values arising from the form factor fit. The upturn in intensity at low q in the SAXS profile for βAβAKLVFF may reflect intercylinder interactions within the larger-scale twisted ribbon superstructure of the fibrils. The SAXS data for βAβAKLVFF-PEG, after enzymatic cleavage, can be interpreted as a complex superposition of profiles from βAβAKLVF and the remaining RC (this can be seen in the high-q part of the SAXS data, which follows closely the form factor of the enzyme) plus a background of F-PEG3000 in aqueous solution. The SAXS data from βAβAKLVF could not be fitted to a form factor related to fibrils, as expected on the basis of the cryo-TEM data. FTIR spectroscopy was used to examine changes in the secondary structure upon enzymatic cleavage via analysis of the amide I region, which contains features dependent on the carbonyl stretch, influenced by the amide hydrogen bonding pattern. The spectrum for βAβAKLVFF-PEG in D2O reveals a peak at 1635 cm-1 (Figure 3). This increases in intensity with increasing concentration. This peak is assigned to β-sheet structure.29 Spectra for βAβAKLVFF are included for comparison. The (27) Hamill, A. C.; Wang, S.-C.; Lee, C. T. Biochemistry 2007, 46, 7694–7704. (28) Serpell, L. C. Biochim. Biophys. Acta 2000, 1502, 16–30. (29) Stuart, B. Biological Applications of Infrared Spectroscopy. Wiley: Chichester, U.K., 1997.
11626 DOI: 10.1021/la101806z
several concentrations in D2O (from top to bottom: 24, 14, 11, and 1.8 wt %), with spectra (lower three) for solutions of βAβAKLVFF at concentrations (top to bottom)of 11, 5, and 1.8 wt % included for comparison.
strong peak at 1672 cm-1 is due to residual TFA.30,31 There is an additional broad peak centered at 1640 cm-1 that is assigned to random coil structure.21 To investigate β-sheet ordering, several higher concentrations were studied. The corresponding spectra for solutions containing 5 or 11 wt % peptide contain a peak at 1627 cm-1 that is the expected position for the main β-sheet peak (there is a shoulder peak near 1612 cm-1).21 This confirms the development of β-sheet ordering at sufficiently high concentration. There is a notable difference in the β-sheet features for βAβAKLVFF and the PEGylated conjugate. The latter shows only a peak close 1635 cm-1, which is also in the range observed for β-sheet structures. The absence of a peak in the 1680 - 1690 cm-1 range is consistent with a parallel rather than an antiparallel β-sheet structure for the PEGylated conjugate, as expected from the constraint imposed by the attached PEG chain.18,19 In contrast to βAβAKLVFF and βAβAKLVFF-PEG, peptide βAβAKLVF lacks β-sheet structure (Figure S8a), consistent with the absence of fibrils by TEM. Circular dichroism confirms that substantial conformational changes occur when βAβAKLVFF-PEG is enzymatically degraded in the presence of RC (Figure 4). The CD spectrum for a 0.5 wt % solution of βAβAKLVFF-PEG exhibits features that indicate the absence of well-defined secondary structure. The weak maximum centered at around 219 nm may be due to a Cotton effect, or we have suggested previously that it may contain a contribution from aromatic stacking interactions based on the observation of similar spectra for FFKLVFF-PEG.18,19 The CD spectrum for a 0.5 wt % solution of βAβAKLVFF alone shows maxima at 212 and 217 nm and resembles our previous results for the same sample in a more concentrated solution (1 wt %).21 The CD spectrum for R-chymotrypsin (Figure 4a) resembles that previously reported,32,33 having a strong minimum at 203 nm and a subsidiary minimum at 230 nm. Upon addition of RC to a solution of βAβAKLVFF-PEG, dramatic changes in the CD spectrum are observed, with a large decrease in ellipticity compared to that of the enzyme alone (Figure 4). The CD spectrum of the βAβAKLVFF-PEG þ RC system is relatively stable as a function of time following the addition of the enzyme. The spectrum of βAβAKLVF (Figure S8b), which is the main fragment (30) Gaussier, H.; Morency, H.; Lavoie, M. C.; Subirade, M. Appl. Environ. Microbiol. 2002, 68, 4803–4808. (31) Pelton, J. T.; McLean, L. R. Anal. Biochem. 2000, 277, 167–176. (32) Manavalan, P.; Johnson, W. C. Nature (London) 1983, 305, 831–832. (33) Lahari, C.; Jasti, L. S.; Fadnavis, N. W.; Sontakke, K.; Ingavle, G.; Deokar, S.; Ponrathnam, S. Langmuir 2010, 26, 1096–1106.
Langmuir 2010, 26(14), 11624–11627
Castelletto et al.
Letter
Figure 4. Circular dichroism spectra. (a) Full range of measured spectra. (b) Expanded range, showing (solid curve) the spectrum calculated by the fractional addition of spectra for R-chymotrypsin and βAβAKLVFF. (Blue 9) 0.5 wt % RC; (Δ) 0.5 wt % βAβAKLVFF-PEG; (green 9) 0.5 wt % βAβAKLVFF; (purple 9) 0.5 wt % RC þ 0.5 wt % βAβAKLVFF-PEG, 0 h after mixing; (red )) 0.5 wt % RC þ 0.5 wt % βAβAKLVFF-PEG, 17 h after mixing; (red b) 0.5 wt % RC þ 0.5 wt % βAβAKLVFF-PEG, 48 h after mixing.
produced by enzymatic degradation, is qualitatively similar to that of βAβAKLVFF, pointing to the lack of sensitivity of CD to the β-sheet structure under these conditions for this system. The spectra after enzymatic cleavage have a maximum closer to the position of the maximum observed for βAβAKLVFF (and βAβAKLVF) than for the PEGylated conjugate, for which the maximum is at somewhat longer wavelength. This is consistent with the presence of a mixture of peptide and enzyme in the system. In summary, cryo-TEM and SAXS indicate that βAβAKLVFF-PEG (containing PEG3000) forms spherical micelles. This first establishes that conjugation to PEG completely disrupts the helically twisted ribbon structure adopted by the parent peptide. Enzymatic cleavage of the peptide-PEG conjugate has successfully been carried out, releasing peptide βAβAKLVF that is shown not to self-assemble into fibrils. We suggest that this is consistent with the important role of phenylalanine in driving the self-assembly process34 because it appears that two terminal F residues are required in order for fibrils to develop. It seems likely that the presence of two β-alanine residues also contributes to the absence of β-sheet structure in the released peptides. These residues cannot participate in the β-sheet hydrogen-bonding pattern because this was actually part of the design criterion for this peptide, which is under study as a fibrillization or oligomerization inhibitor. Our work shows that it is possible to design a delivery system that can release amyloid peptide fragments containing a self-recognition element, in response to an enzymatic (34) Gazit, E. FEBS J. 2005, 272, 5971–5978.
Langmuir 2010, 26(14), 11624–11627
trigger. In future work, it would be interesting, for example, to investigate systems responsive to the β- and γ-secretase enzymes that release Aβ from the amyloid precursor protein (APP).35 We envisage that our work will stimulate the development of peptidePEG nanocontainer systems, in a variety of directions, toward enzyme-responsive diagnostics or therapeutics. Acknowledgment. This work was supported by EPSRC grants EP/F048114/1 and EP/G026203/1 to I.W.H. We are grateful to Ge Cheng for synthesizing the βAβAKLVF peptide. Use of the Chemical Analysis Facility at the University of Reading is acknowledged. Supporting Information Available: Materials. Experimental Details: Mass spectrometry, cryo-TEM, SAXS, FTIR, CD, and pyrene fluorescence. Electrospray mass spectrum after treatment of βAβAKLVFF-PEG with RC. MS/MS spectrum for peptide isolated by enzymatic cleavage. MS/ MS spectrum of synthetic βAβAKLVF. Cryo-TEM image of an aqueous 1.8 wt % solution of βAβAKLVFF. Negative stain TEM of a βAβAKLVF solution. Representative cryoTEM images from an aqueous solution containing 0.85 wt % PEG3500 þ 0.18 wt % βAβAKLVF. Pyrene fluorescence determination of the cmc. Spectroscopy data for solutions of βAβAKLVF. Parameters used to model the SAXS curves in Figure 2. This material is available free of charge via the Internet at http://pubs.acs.org. (35) Hamley, I. W. Angew. Chem., Int. Ed. 2007, 46, 8128–8147.
DOI: 10.1021/la101806z
11627
