Biomacromolecules 2008, 9, 2997–3003
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Vesicles from Peptidic Side-Chain Polymers Synthesized by Atom Transfer Radical Polymerization Dave J. Adams,*,†,‡ Derek Atkins,§ Andrew I. Cooper,| Steve Furzeland,§ Abbie Trewin,| and Iain Young*,‡ Unilever Corporate Research and Unilever R&D Colworth, Sharnbrook, Bedford, MK44 1LQ, United Kingdom, and Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, United Kingdom Received January 29, 2008; Revised Manuscript Received August 15, 2008
Block copolymers can adopt a wide range of morphologies in dilute aqueous solution. There is a significant amount of interest in the use of block copolymer vesicles for a number of applications. We show that a series of oligo(valine) and oligo(phenylalanine) peptides coupled to a methacrylic group can be prepared by conventional peptide coupling techniques. These can be successfully polymerized by atom transfer radical polymerization (ATRP) in hexafluoroisopropanol (HFIP) giving access to poly(ethylene oxide)-b-poly(side-chain peptides). Many of these polymers self-assemble to form vesicles using an organic to aqueous solvent exchange. One example with a divaline hydrophobic block gives a mixture of toroids and vesicles. Circular dichroism demonstrates that secondary structuring is observed in the hydrophobic region of the vesicle walls for the valine side-chain containing polymers.
1. Introduction Block copolymers can adopt a wide range of morphologies in dilute aqueous solution.1,2 The morphologies adopted depend on many factors including the block compositions and the preparation conditions.3,4 Currently, there is a significant amount of interest in the use of block copolymer vesicles for the encapsulation of hydrophilic materials.5-14 The majority of the work utilizes synthetic block copolymers such as poly(acrylic acid)-b-poly(styrene)15-17 and poly(ethylene oxide)-b-poly(caprolactone).5,18 Deming et al.,19-21 Lecommandoux et al.,22 and Iatrou et al.23 have recently reported on block copolypeptides and showed that vesicles can be prepared where the peptidic blocks adopt defined secondary structures, incorporating this secondary structure into the hydrophobic domains of the vesicle walls. Vesicles have also been prepared from a number of polymer-peptide conjugates,24-32 although in many cases the peptide block forms the corona rather than the core of the vesicle walls. Polymer-peptide conjugates have been prepared in a number of ways9,33,34 with recent reports describing the use of living radical polymerization techniques such as atom transfer radical polymerization (ATRP). van Hest et al. have reported the synthesis of β-sheet side chain PEO-peptide conjugates by ATRP.35,36 Investigation into the self-assembly of these architectures in solution has been restricted to that of block copolymers with elastin- and silk-like peptide units. It has been shown that such polymers do adopt secondary structures similar to that expected for the oligopeptides making up the side chains. Other PEO-(linear peptide) conjugates have also been shown to adopt structures driven by the peptide block. For example, it has been shown that a PEO-peptide conjugate where the peptide consisted of five diads of alternating threonine and valine residues (with a high propensity to form β-sheets) adopts a tape* To whom correspondence should be addressed. E-mail: d.j.adams@ liverpool.ac.uk;
[email protected]. † Current address: Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, U.K. ‡ Unilever Corporate Research. § Unilever R&D Colworth. | University of Liverpool.
like morphology in solution.37 PEO-(linear peptide) conjugates have been shown to form nanofibers in solution38-40 with these morphologies highly reminiscent of structures adopted by peptides. However, a number of other reports detail the assembly of polymer-peptide conjugates into micelles41-44 or vesicles24-32 as would be expected from conventional amphiphilic block copolymers in dilute aqueous solution. Here, we postulated that it would be possible to exploit the secondary structuring ability of peptides and yet maintain the synthetic simplicity of ATRP to prepare side-chain peptide PEO-peptide conjugates that form vesicles in solution using side-chain peptides similar to those reported by van Hest et al. This could be achieved by tuning the ratio of PEO to peptide as well as the length of the oligopeptide in the side chains. In these cases, the peptide block would form the hydrophobic walls of the vesicle and potentially adopt secondary structure. To investigate this, we have prepared a range of PEO-b-poly(side-chain peptide) copolymers using oligo(valine), expected to adopt a β-sheet conformation in water, and oligo(phenylalanine), expected to adopt an R-helical conformation in water at sufficiently long monomer lengths.45 We demonstrate the synthesis of a family of methacrylicterminated peptides, which can be polymerized using ATRP to give block copolymers and show that these can adopt welldefined self-assembled structures in dilute aqueous solution.
2. Materials and Methods 2.1. Materials. Methoxy-PEO-macroinitiators were prepared as described previously.4 2-Aminoethylmethacrylate hydrochloride was purchased from Aldrich as technical grade and purified by washing with THF. The final purity was >95%, as determined by NMR (see Supporting Information). All other chemicals were purchased from Aldrich and used as received. All peptides were synthesized by the stepwise coupling of N-Boc-protected amino acid to a C-ethyl ester protected amino acid or oligopeptide using isobutylchloroformate in chloroform. Full experimental and characterization data for peptides and monomers used are described in the Supporting Information. 2.2. Characterization. NMR. 1H NMR spectra were recorded at 600 MHz using a Bruker AV(II) 600 NMR spectrometer. Gel Permeation Chromatography. GPC were carried out on a PLGPC 50 equipped with two 5 µm (30 cm) mixed “C” columns and a
10.1021/bm8006693 CCC: $40.75 2008 American Chemical Society Published on Web 09/27/2008
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refractive index detector.4 Molecular weights of the polymers were determined using PL Cirrus GPC software (version 2.0) supplied by Polymer Laboratories. A series of near-monodisperse linear PMMA standards (purchased from Polymer Laboratories) were used to construct the calibration curve using THF as an eluent at a flow rate of 1.0 mL min-1 at 30 °C for PEO-b-poly(side-chain peptide) copolymers. Dynamic Light Scattering. DLS was performed using an ALV laser goniometer, consisting of a 22 mW HeNe linear polarized laser operating at a wavelength of 632.8 nm and an ALV-5000EPP multiple τ digital correlator with 125 ns initial sampling time. Data were acquired using ALV Correlator Control Software, version 3.0.2.3, over a range of angles from 45 to 150°. The sizes quoted were measured at an angle of detection of 90°. Solutions were placed in 10 mm diameter glass cells and maintained at 25.0 ( 0.1 °C. Transmission Electron Microscopy. Samples for thin-film cryo-TEM were prepared using a GATAN cryo-plunge into liquid ethane and then transferred using a CT3500 cryo-transfer system. Samples were examined using a JEOL 1200 EX TEM operating at 100 kV. Images were obtained using a Bioscan CCD camera and processed using the Gatan Digital Micrograph software. Wall thicknesses were measured using this software. Circular Dichroism. Circular dichroism was carried out on an OLIS DSM CD with a 1 mm path length glass sample cell in the range from 200 to 270 nm. The subtracted background spectrum (water) consisted of an average of five scans. An average of five scans was also used for each sample. 2.3. Polymerization of Peptide-Functionalized Methacrylates. The methoxy poly(ethylene oxide)2k ATRP macroinitiator (0.14 g, 0.065 mmol), methacrylate functionalized peptide (20 mol equiv), and 2,2′bipyridine (20 mg, 0.13 mmol) were degassed for 30 min and placed under nitrogen. Propan-2-ol or hexafluoropropan-2-ol was added (2 mL) and heated to 55 °C. Copper bromide (10 mg, 0.065 mmol) was then added quickly to the stirred solution to give a final ratio of mPEO/ methacrylate/CuBr/bipy of 1:20:1:2. The reaction color changed to a deep red-brown color and the solution quickly viscosified. After 18 h, tetrahydrofuran was added to the reaction mixture (∼5 mL) and the solution changed to a blue-green color. This was filtered through basic alumina to remove the copper residues and then dried in vacuo to yield a crispy off-white solid. These were further purified by extensive dialysis in deionized water from a tetrahydrofuran:water (2:8) solution using 12K MWCO dialysis tubing at room temperature, changing the external water at least eight times over 2 days. The dialyzed product was freeze-dried to obtain a fine white powder. Yields were between 90 and 99%. The final purities were >95% as determined by 1H NMR. 2.4. Preparation of Micellar Solutions. Aqueous dispersions of block-copolymer aggregates were prepared by dissolving the polymer in THF to a concentration of 5 mg mL-1. Millipore water (prefiltered through a 0.2 µm PVDF filter) was added manually to give a final polymer concentration of 1 mg mL-1. The solvent was then removed by dialysis against 5 L of water using 12K MWCO dialysis tubing at room temperature, changing the external water at least eight times over 2 days. Final polymer concentrations after dialysis were not significantly changed. Cryo-TEM was carried out on samples at this concentration. DLS measurements were carried out at this concentration and on samples diluted by an order of magnitude to ensure the absence of multiple scattering. CD measurements were carried out on solutions diluted to a concentration of 0.5 mg mL-1. 2.5. Atomistic Simulations. Molecular models for peptide chains were generated using the Materials Studio Modeling 4.0 package (Accelrys Inc., San Diego, CA, 2005). The aliphatic and phenyl peptide chains were constructed using the polymer build tool with 15 repeat monomer units per chain. The interactions between adjacent peptide chains were investigated by constructing two models for each system containing three parallel peptide chains: first, placing the peptide chains within close proximity with a backbone separation of 1.0 nm, and second, a greater backbone separation of 1.5 nm. All models were fully relaxed using the Discover molecular mechanics and dynamics simula-
Adams et al. Scheme 1. Synthesis of a Bis-protected Dipeptide by Coupling an N-Boc-protected Amino Acid with a C-Ethyl Ester Protected Amino Acida
a
IBCF, iso-butylchloroformate; NMM, N-methylmorpholine.
Scheme 2. Synthesis of Peptide-Functionalized Methacrylates by Coupling of a N-Boc-protected Amino Acid with 2-Aminoethylmethacrylatea
a
IBCF, iso-butylchloroformate; NMM, N-methylmorpholine.
tion module with the COMPASS forcefield.46,47 For the aliphatic peptide, an average chain separation of 1.1 nm was found to be favorable with the peptide side groups interpenetrating to a large extent. Significant hydrogen bonding is observed, both within the peptide chain and between separate peptide chains, whereas for both poly(F2) and poly(F3) peptide chains, an average separation of 1.6 nm is favored with less interpenetration of the peptide side groups.
3. Results 3.1. Monomer Preparation. A series of peptide-functionalized methacrylates have been prepared using standard peptide coupling techniques.48 The oligopeptides were first prepared by sequential coupling of N-Boc-protected amino acids to a C-ethyl ester protected amino acid or oligo(peptide) using isobutylchloroformate in chloroform, Scheme 1. Using this methodology, a series of oligo(L-valine) and oligo(L-phenylalanine) polymers were prepared. These will be referred to as Vx and Fy, where x and y represent the number of either valine or phenylalanine units joined via peptide linkages (i.e., V1 and F1 are the amino acids, V2 and F2 the dipeptides, and V3 and F3, the tripeptides). Boc-protected amino acids were sequentially added to the C-protected peptide, followed by removal of the Boc protecting group using trifluoroacetic acid. Each of these steps was found to be high yielding. It was found to be significantly easier to follow this procedure rather than remove the ethyl ester protecting group from the C-terminus and attempt linking two oligopeptides. Here, yields were significantly lower and the purity was harder to control, with incomplete deprotection of the C-terminus leading to mixtures from which separation of the required peptides were found to be extremely difficult. After building to the required length, the ethyl ester protecting group was then removed. Here, lithium hydroxide in a THF/ water solvent mixture was used. The oligopeptide was then coupled to the N-terminus of 2-aminoethylmethacrylate, Scheme 2. 2-Aminoethylmethacrylate hydrochloride is currently only commercially available in technical grade (∼ 90% pure). Although a synthetic strategy has been published,49 it was possible to purify the commercial material by washing with THF. The hydrochloride salt is insoluble in cold THF and, hence, can be washed until all impurities are removed and can routinely be collected in greater than 95% purity (see Supporting
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Scheme 3. Synthesis of PEO-b-poly(side-chain peptide) Copolymers by ATRP
Table 1. GPC Data for the PEO-b-poly(side-chain peptide) Copolymers (in THF)a monomer
Mw (g mol-1)
Mn (g mol-1)
Mw/Mn
d.p.b
Mn (g mol-1)c
wt % PEOd
V1 V2 V3 F1 F2 F3
10800 8900 10900 11000 8400 8200
9600 7900 8500 9800 7600 7300
1.13 1.12 1.29 1.12 1.11 1.12
21 25 7 18 22 8
8900 12600 5700 8750 13400 7300
23 16 35 23 15 34
a All molecular weight data was calculated by comparison to PMMA standards. b Degree of polymerization (d.p.) determined by NMR. In all cases, a degree of polymerization of 20 was targeted. c Data obtained from 1H NMR. d Weight percentage of PEO in the block copolymer calculated from the d.p. determined by 1H NMR.
Information). Coupling the oligopeptides to the N-terminus of 2-aminoethylmethacrylate was found to be efficient. 3.2. Polymerization of Peptidic Monomers. Polymerization of these peptide-functionalized methacrylates was carried out by ATRP following published procedures.50 PEO-based macroinitiators were used to prepare a series of PEO-b-poly(sidechain peptide) copolymers, Scheme 3. Initial experiments using the V1 and F1 monomers were successful in propan-2-ol. However, using longer peptidic monomers led to the reaction mixtures becoming blue after a very short time. Using HFIP, a much better solvent for both oligopeptides and polypeptides,51 as solvent for the reaction resulted in successful polymerization taking place. Polymerization was found to be more sluggish for both the V3 and F3 monomer, with the final degree of polymerization being significantly lower in these cases. After synthesis, residual monomer and solvents were removed by extensive dialysis against water. An example NMR spectrum of a protected PEOb-poly(side-chain peptide) is shown in Figure 1. Molecular weight data for the N-Boc-protected polymers is detailed in Table 1.
Figure 1. 1H NMR spectrum of a PEO-b-poly(side-chain peptide) copolymer. The polymer shown is PEO-b-poly(V1). Included is the assignment of the polymer. The peaks marked “X” arise from the solvent (DMSO-d6) and HOD present in the solvent.
Table 2. DLS Data for the PEO-b-poly(side-chain peptide) Copolymers at a Polymer Concentration of 1 mg mL-1a monomer V1 V2 V3 F1 F2 F3
Rh/nm 81 ( 68 ( n.d. 84 ( 59 ( 63 (
2 15 5 12 9
PDI 0.22 0.14 n.d. 0.15 0.32 0.18
( 0.07 ( 0.03 ( 0.02 ( 0.12 ( 0.06
a n.d., not determined due to precipitation on dialysis. Rh and PDI determined from the Cumulants analysis of the correlogram obtained by DLS as an average of three independent measurements.
3.3. Self-Assembly of PEO-b-Poly(side-chain peptide) Copolymers. Amphiphilic block copolymers are known to selfassemble in dilute solution. The self-assembly of such polymers can be carried in a number of ways. When the aqueous solubility of the polymers is low, it is common to initially dissolve the polymer in a water-miscible organic solvent. Water is then added to this solution. The overall solvent quality decreases for the hydrophobic block as water is added, inducing aggregation and, hence, self-assembly of the block copolymer. Finally, the organic solvent is removed by some means, often dialysis. The PEOb-poly(side-chain peptide) copolymers were found to be too insoluble in water by simple rehydration techniques. Hence, the polymers were first dissolved in THF at a concentration of 5 mg mL-1. Deionized water was then added to give a final polymer concentration of 1 mg mL-1. The THF was then removed by extensive dialysis against water. Several characterization techniques were employed to ascertain the nature of the self-assembled structures in solution including dynamic light scattering (DLS), circular dichroism (CD), and cryo-TEM. The DLS data shows that the Boc-protected polymers produce relatively monodisperse (polydispersity
