Macromolecules 2005, 38, 761-769
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PEG-Based Hybrid Block Copolymers Containing R-Helical Coiled Coil Peptide Sequences: Control of Self-Assembly and Preliminary Biological Evaluation Guido W. M. Vandermeulen,† Christos Tziatzios,‡ Ruth Duncan,§ and Harm-Anton Klok*,⊥ Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany; Johann Wolfgang Goethe-Universita¨ t, Institut fu¨ r Biophysik, Theodor-Stern-Kai, D-60590 Frankfurt am Main, Germany; Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK; and E Ä cole Polytechnique Fe´ de´ rale de Lausanne, Institut des Mate´ riaux, Laboratoire des Polyme` res, Baˆ timent MX-D, CH-1015 Lausanne, Switzerland Received July 15, 2004; Revised Manuscript Received November 10, 2004
ABSTRACT: This study investigates the structure and organization of a series of hybrid diblock copolymers based on poly(ethylene glycol) (PEG) and peptide sequences inspired by the coiled coil protein folding motif. Circular dichroism spectroscopy and analytical ultracentrifugation experiments indicate that the peptide sequences in these block copolymers act as structure-directing auxiliaries and mediate the formation of discrete nanosized assemblies. This self-assembly process is driven by the very specific folding and organization properties of the peptide sequences and does not involve unspecific interactions leading to large polydisperse structures. The present study also demonstrates that the self-assembly properties of the hybrid block copolymers can be controlled via selective replacement of one or two amino acid residues in the peptide block. This is an attractive feature in view of possible biomedical applications. Preliminary biological experiments show that the properties of these polymers correlate with their selfassembly behavior.
Introduction In biology, inter- and intramolecular association of peptide chains is commonly referred to as oligomerization. Oligomerization can occur between identical peptide sequences (homooligomerization) as well as between different peptide sequences (heterooligomerization). Homo- and heterooligomerization of peptide chains plays an important role in nature where it is involved in protein stability and recognition processes. An excellent example in this regard is the coiled coil protein folding motif, which is a highly versatile tertiary structure identified in more than 200 native proteins.1 Coiled coils consist of 2-5 R-helical peptide strands that are wrapped around each other in superhelical fashion.2 Influenza hemagglutinin (HA),3 the macrophage scavenger receptor,4 and β-lactoglobulin5 are three examples of proteins containing a coiled coil domain, which is of crucial importance to their function. Because of a high net negative charge, the coiled coil domain of these proteins is largely unfolded at neutral pH and folds into a homooligomeric coiled coil at lower pH. The formation of these homooligomers leads to influenza HA’s fusogenicity, macrophage scavenger receptor ligand dissociation and receptor recycling, and β-lactoglobulin’s survival of function at stomach pH. In contrast to the three previous examples, which contain homooligomeric coiled coil motifs, the yeast protein GCN4, which is a transcription factor, contains †
Max Planck Institute for Polymer Research. Johann Wolfgang Goethe-Universita¨t. § Cardiff University. ⊥ E Ä cole Polytechnique Fe´de´rale de Lausanne. * To whom correspondence should be addressed: e-mail
[email protected]; Ph ++ 41 21 693 4866; Fax ++ 41 21 693 5650. ‡
short coiled coil sequences that are able to form heterodimers between an acidic and basic peptide chain.6 This highly specific dimerization process allows the recognition of specific DNA sequences by an adjacent region rich in basic residues and regulation of transcription. The interactions involved in specific heterodimerization have been thoroughly investigated and successfully exploited in the design of de novo heteromeric coiled coils in a number of laboratories.7 Previously, we have shown that the highly specific folding and organization properties of coiled coil peptide sequences are retained upon conjugation to a synthetic polymer, such as for instance poly(ethylene glycol) (PEG).8,9 Dilute solution self-assembly of such PEGylated coiled coil hybrid diblock copolymers is driven by the intrinsic propensity of the peptide blocks to fold into well-defined tertiary structural motifs and can lead to essentially monodisperse supramolecular aggregates.10 We also reasoned that the self-assembly properties of these hybrid block copolymers may be precisely controlled via directed replacement of one or a few amino acids. The aim of the present study is to demonstrate that such substitutions in the peptide’s primary structure can indeed be used to control the degree of oligomerization of PEG-b-peptide diblock copolymers and the pH sensitivity of the resulting aggregates. In addition, we will give a preliminary account of the biological properties of these hybrid diblock copolymers. Experimental Section Materials. Amino acids, resins, O-benzotriazole-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU), and Nhydroxybenzotriazole (HOBt) were purchased from CalbiochemNovabiochem GmbH (Schwalbach, Germany). N-Methyl-2-pyrrolidinone (NMP) was obtained from BASF AG (Ludwigshafen,
10.1021/ma0485538 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/11/2005
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Germany). Murine melanoma B16 F10 cells were a kind gift from Prof. I. Hart (St. Thomas Hospital, London, UK). Male Wistar rats were supplied by Banton and Kingman (Hull, UK). RPMI-1640 and foetal bovine serum (FBS) were purchased from Gibco BRL (Paisley, UK). 3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT), optical grade dimethyl sulfoxide (DMSO), dextran (Mw ) 72 000 g/mol), poly(Llysine) (PLL) (Mw ) 52 100 g/mol), and poly(ethylene imine) (PEI) (Mw ) 70 000 g/mol) were obtained from Sigma Chemicals (Dorset, UK). NMP and N,N-dimethylformamide (DMF) were dried over molecular sieves (4 Å) prior to use. Dichloromethane (DCM) was freshly distilled from P2O5 before use. All other chemicals and reagents were acquired from Sigma Aldrich Chemie GmbH (Deisenhofen, Germany) and were used as received. The synthesis of the PEG carboxylic acid derivatives (mPEG-COOH) was carried out according to a literature procedure.11 Spectra/Por dialysis bags (made from regenerated cellulose) were purchased from Carl-Roth GmbH (Karlsruhe, Germany). MALDI-TOF Mass Spectrometry. Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectra were recorded on a Bruker Reflex II MALDI-TOF mass spectrometer. Samples were dissolved in tetrahydrofuran and mixed with the matrix 2,5-dihydroxybenzoic acid (DHB). Sodium trifluoroacetate was added to facilitate ionization. Reversed-Phase High-Pressure Liquid Chromatography (RP-HPLC). RP-HPLC was performed on a Jasco System (pump/degasser PU-2089+; detector UV-2075+; autosampler AS-2055+; column oven CO 2060+), using a Macherey Nagel C18 reversed-phase column (EC 250/4 NUCLEOSIL, 500 Å pore size, 5 µm particle size). Samples were eluted with a linear AB gradient from A to B over 20 min, with A consisting of 80/20 (v/v) water/acetonitrile (plus 0.1% TFA) and B of 100% acetonitrile (plus 0.1% TFA). The flow rate was 0.7 mL/min. Sample elution was monitored with a UV/vis detector at 210 nm. In all cases the purity of the peptides and the diblock copolymers was found to be g95%. UV/vis Spectroscopy. UV/vis spectra were recorded on a Perkin-Elmer Lambda 15 spectrophotometer. Molar extinction coefficients were determined by absorption measurement of samples with known concentration and deviated less than 5% from the molar exctinction coefficient of tyrosine (Tyr) reported in the literature (280 ) 1197 M-1 cm-1).12 Therefore, the literature value of the molar extinction coefficient of Tyr was used for determining sample concentrations. Concentrations were calculated according to Lambert-Beer’s law. Circular Dichroism. Circular dichroism (CD) spectra were recorded on a Jasco J-715 spectropolarimeter equipped with a Jasco PTC-348WI temperature-controlled cell. Details of the CD experiments have been published before.8 For pH-dependent experiments, a stock solution in phosphate buffered saline (PBS, consisting of 10 mM sodium phosphate, 140 mM NaCl, 3 mM KCl) at pH 7.4 was prepared. The pH of the solution was adjusted using either a 0.1 M HCl or NaOH solution until the desired pH was obtained. The sample solutions were kept at room temperature for at least 15 h before measurement. The pH did not change significantly after the 15 h incubation time ((0.1 pH units). All CD spectra at different pH were carried out at a concentration of ∼100 µM. Analytical Ultracentrifugation (AUC).13 Sedimentation equilibrium experiments were performed with a Beckman Optima XL-A analytical ultracentrifuge using Epon 6-channel centerpieces with a path length of 1.2 cm in combination with an An-60Ti rotor. Details of the AUC experiments have been published before.8 Samples were prepared as described above for the pH-dependent CD experiments. Peptide and PEG-b-peptide Synthesis. Peptide and PEG-b-peptide synthesis and purification were performed as described before.8 Typically, after a 0.25 mmol scale synthesis, 90 mg (14%) of pure peptide was obtained as a white powder. Diblock copolymers were obtained as white powders in about 25% yield. Determination of IC50 Values. Adherent murine melanoma B16 F10 cells were maintained in RPMI 1640 medium, with 5 mM L-glutamine and 10% (v/v) FBS, and cultured as a
Macromolecules, Vol. 38, No. 3, 2005 monolayer in a humidified atmosphere containing 5% CO2 at 37 °C and neutral pH. Cells were seeded to a sterile, 96-well plate at a seeding density of 1 × 103 cells per well and allowed to grow for 24 h under standard conditions. After removal of the media, sample solutions in RPMI 1640 medium were added in a concentration range of 0-5 mg/mL. The cultures were incubated under standard conditions for 67 h. After the 67 h incubation, cell viability was assessed using the MTT assay.14 First, 20 µL of a 5 mg/mL solution of MTT in PBS (pH 7.4) was added to each well and further incubated under standard conditions for 5 h. After this period, the culture medium was removed and 120 µL of DMSO was added. The plates were read at 550 nm using a microtiter plate reader (Sunrise Tecan Plate Reader). The results were expressed as cell viability (%) relative to a control containing no sample. The reported values represent the means ((sem) of three experiments, each containing six replicates. IC50 values were calculated using regression analysis for each of the separate experiments, and the mean ((sem) values are shown. Dextran and PLL were used as nontoxic and toxic controls, respectively. Haemolysis Assay.14,15 Male Wistar rats (∼250 g body weight) were killed by CO2 asphyxiation, and blood was obtained by cardiac puncture. A 2% (w/v) suspension of red blood cells (RBCs) was made as follows. First, the blood was centrifuged at 1000 rpm at 4 °C for 10 min, and the plasma and top 2-3 mm of the pellet were then removed and discarded. The pellet was resuspended in PBS (pH 7.4) that had been prechilled to 4 °C. The suspension was then subjected to another centrifugation step, as described before. This process was repeated one more time, and the pellet was resuspended in a volume appropriate to 2% (w/v) suspension using PBS pH 7.4. Samples were dissolved at concentrations ranging from 0 to 1 mg/mL in PBS and added to an equal volume of 2% (w/v) RBC suspension in PBS using 96-well plates. This solution was left for 1 h at 37 °C. After the incubation period, the 96-well plates were subjected to centrifugation at 1500 rpm for 15 min. The supernatant was then transferred to a new 96-well plate. Dextran and PEI were used as reference controls, and Triton X-100 (1% v/v) was used to give a 100% haemolysis value. Hemoglobin release was determined spectrophotometrically at 550 nm and expressed as a percentage of hemoglobin release relative to the Triton X-100 control. DNA Complexation and Gel Electrophoresis. The interaction between DNA and X-KKK was investigated by electrophoresis on an agarose gel. A solution containing EcoR1cleaved plasmid pSV-β-galactosidase in 10 mM Tris‚HCl, pH 8.5 (1.3 µL, 0.40 mg/mL), was added to the appropriate X-KKK solution in PBS (pH 7.4). Sterile, double distilled water was added to a total volume of 15 µL. Complexes were prepared at a polymer:DNA molar ratio of 50:1, 100:1, 200:1, 400:1, 700: 1, and 1000:1. The samples were then pulse-spun at 13 000 rpm in a microcentrifuge and incubated at room temperature for 30 min. Loading buffer (5 µL of 0.25% w/v bromophenol blue in 40% glycerol/60% tris-borate-EDTA (TBE) buffer) was added, and the samples were respun. Samples were then loaded into a 0.8% (w/v) agarose gel containing ethidium bromide (0.1 µg/mL). Controls for free DNA, peptide, and block copolymers were also applied to the gel. Gel running buffer was 0.25x TBE (adjusted to pH 8.3 with glacial acetic acid). The gel was run at 100V for 60 min, after which the DNA was visualized on an UV transilluminator. Gels were photographed using a Polaroid gel-documenting system.
Results and Discussion Design and Synthesis. The composition and chain length of the peptide segment of the diblock copolymers are identical to that of our previously published coiled coil sequence (heptad repeat “a-g”: LAEIEAK).8 The heptad repeat of the newly designed series, however, contains L-isoleucine in position a and L-leucine in position d (IAELEAK). On the basis of literature date, this replacement would most likely result in the forma-
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Table 1. Amino Acid Sequence and Composition of the Investigated Peptides and Block Copolymers
tion of a two-stranded R-helical coiled coil.16-18 LGlutamic acid and L-lysine residues in positions e and g, respectively, can form interhelical salt bridges between the peptide chains and promote a parallel orientation.7b L-Alanine is chosen in positions b and f because of its high intrinsic helix propensity, and L-glutamic acid is placed in position c because of possible stabilizing intrahelical electrostatic interactions with L-lysine in position g (i, i + 4 interaction). Glycine is introduced at the N-terminus to reduce steric hindrance for PEG coupling and improve the coupling yield of a PEG chain.19 Finally, the presence of L-tyrosine at the C-terminus enables accurate determination of sample concentration by means of UV/vis spectroscopy. To control the pH sensitivity of the coiled coil domains and allow pH-induced folding and unfolding, two analogues of the IAELEAK sequence were designed, which contain either L-glutamic acid instead of L-lysine at position g (IAELEAE) or L-lysine instead of L-glutamic acid at positions c and e (IAKLKAK). The rationale behind the design of these analogues was to eliminate attractive electrostatic interactions at neutral pH and induce folding and association under acidic (IAELEAE) or basic (IAKLKAK) conditions, respectively. In addition, mixing equal amounts of the complementary IAELEAE- and IAKLKAK-based peptides or hybrid block copolymers is anticipated to result in a novel heteromeric coiled coil motif. In the following, the different samples will be designated as X-ceg, where X represents either an acetyl group (Ac) or a mPEG(750) or mPEG(2000) chain and c, e, and g coiled coil heptad repeat positions, which are occupied by either L-glutamic acid (E) or L-lysine (K) residues. Table 1 gives an overview of the amino acid sequence and composition of the peptides and block copolymers that have been investigated. The peptides and block copolymers were prepared using standard Fmoc solid-phase peptide synthesis, following a procedure which has been described in an earlier publication.8 All compounds are characterized by 1H NMR spectroscopy, reversed-phase HPLC, and MALDI-TOF mass spectrometry. HPLC chromatograms and mass spectra for all compounds are given in Figures I and II of the Supporting Information. Self-Organization of X-EEK. At pH 7.4, the peptide and diblock copolymers show R-helix-like CD spectra with negative minima at 222 and 208 nm and a positive maximum at about 195 nm (Figure III in the Supporting Information). From the mean residue ellipticity at 222
Figure 1. Plots showing the concentration dependence of (a) the helix content and (b) [θ]222/[θ]208 for X-EEK in PBS at pH 7.4.
nm, the helix content of the peptide segment can be calculated.20,21 As shown in Figure 1, Ac-EEK has a high (>90%) and concentration-independent helix content at pH 7.4 over the entire concentration range studied (10210 µM), whereas mPEG(750)-EEK and mPEG(2000)EEK show a slightly concentration-dependent helix content, i.e., from ∼73% at 10 µM to ∼90% at 260 µM. These CD spectra provide a first hint that the folding and organization properties of the peptide sequences are retained after conjugation of PEG and that they drive the self-assembly of the block copolymers into discrete nanosized aggregates. As we have shown previously, the slightly lower helix content for the diblock copolymers compared with the peptide reflects the smaller percentage of folded assemblies present in solutions of these samples due to the steric hindrance of the PEG chain, which hampers oligomerization of the block copolymers.8 The ellipticity ratio [θ]222/[θ]208 is virtually concentration-independent for all three samples and has a value of 0.97-1.02, which is typical for coiled coil tertiary structures.22 While the CD experiments point toward the formation of coiled coil aggregates, they neither provide information about the aggregation number of these superstructures nor exclude the possible formation of large unde-
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Table 2. Unimer (U), Dimer (D), Tetramer (T), and Higher Oligomer (HO) Content (wt %) of X-EEK, X-EEE, X-KKK, and Equimolar Mixtures of X-EEE and X-KKK at Different pH Values, Estimated from Sedimentation Equilibrium Analysis Assuming a Unimer/Dimer/Tetramer Association Modela pH 3.7 Ac-EEK mPEG(750)-EEK mPEG(2000)-EEK Ac-EEE mPEG(750)-EEE mPEG(2000)-EEE Ac-KKK mPEG(750)-KKK mPEG(2000)-KKK heteromeric peptide heteromeric mPEG(750)-conjugate heteromeric mPEG(2000)-conjugate
U
D
21 30 10 28 -
62 35 34 97
pH 7.4
T
HO
17 56 72 3
35 -
b
-
-
28
41
b
pH 9.8
U
D
T
HO
85 55 80 10 15 45
5 5 5 23 20
8
13 34c 81
17
47
-
-
15 32 15 95 90 85 95 77 35 66 11
30
1
36
U
D
T
HO
28 30
62 70
10 -
-
55 22
45 78
-
-
-
-
-
-
-
-
43
17
34
6
b
b
a All experiments were carried out at a sample concentration of 100 µM. b It was not possible to determine the precise amounts of the different oligomers. For Ac-EEK and the heteromeric peptide, however, the average molecular weight was independent of the pH (see Figures V and XII of the Supporting Information), and the formation of large aggregates via unspecific association can be excluded. c It was not possible to determine the exact identity and concentration of the small oligomers present. The presence of around 66% unimers is, however, relatively sure.
fined structures via unspecific association processes. To this end, the X-EEK series was studied with AUC (Table 2). Sedimentation equilibrium analysis at pH 7.4 and a concentration of 100 µM indicates that Ac-EEK predominately (85 wt %) forms dimers, while the remainder consist of unimers. Since the amphiphilic peptide chains are largely unfolded in their unimeric state, the low concentration of unimeric Ac-EEK measured by AUC is in agreement with the very high helix content observed in the CD experiments. The major component of both mPEG(750)-EEK and mPEG(2000)-EEK at pH 7.4 and a concentration of 100 µM is a dimeric assembly, ∼55 and ∼80 wt %, respectively. In addition, mPEG(750)EEK also contains ∼32 wt % unimers and ∼13 wt % tetramers, whereas mPEG(2000)-EEK also contains ∼15 wt % unimers and ∼5 wt % trimers or hexamers. Together with the CD experiments, the AUC data prove that the EEK sequence exclusively mediates the formation of small, discrete-sized aggregates and does not induce the formation of large aggregates via unspecific interactions. Also after conjugation of PEG, the peptide sequences retain their preference to preferably form dimeric aggregates, although the specificity in the case of the diblock copolymers is slightly lower in comparion with the Ac-EEK peptide. Figure 2 shows plots of the pH dependence of the helix content and the mean residue ellipticity ratio [θ]222/[θ]208 for the X-EEK series at a concentration of 100 µM (for CD spectra, see Figure IV in the Supporting Information). The helix content of Ac-EEK is nearly constant between pH 7 and 11: a decrease and increase occurring only above pH 11 and below pH 7, respectively. Below ∼pH 4.4, which is the pKa value for L-glutamic acid, the peptide starts to precipitate. The ellipticity ratio [θ]222/ [θ]208 remains 1.00 for Ac-EEK over the entire pH range, and according to AUC experiments, the average molecular weight of the Ac-EEK sample is virtually independent of pH (5.15-5.55 kDa between pH 3.7 and pH 9.8; Figure V in the Supporting Information), which supports the formation of predominately dimeric Ac-EEK assemblies (MW ) 5.0 kDa). In contrast to Ac-EEK, the corresponding block copolymers are soluble over the entire pH range (pH 1-12). Between pH 4.4 and 10.2, which are the pKa values of L-glutamic acid and L-lysine, respectively, intermolecular (i, i′ + 2) and intramolecu-
Figure 2. Plots showing the pH dependence of (a) the helix content and (b) [θ]222/[θ]208 for X-EEK (PBS, 100 µM).
lar (i, i + 4) electrostatic interactions stabilize the R-helical secondary structure and the coiled coil superstructure, and the helix content and [θ]222/[θ]208 are
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independent of pH. Below pH 4.4 and above pH 10.2, stabilizing Glu-Lys salt bridges between the charged residues at e and g positions are lost, which may explain the observed decrease in helix content. The ellipticity ratio of ∼1.0 above pH 10.2 suggests that the coiled coil superstructure is preserved in this pH regime, presumably due to stabilizing inter- and intramolecular hydrogen-bonding interactions. Below pH 4.2, however, the ellipticity ratio increases to ∼1.1, which may indicate a distortion of the coiled coil superstructure. It has been shown that protonated L-glutamic acid residues in position e can contribute to the hydrophobic core.23 Thus, the stabilizing electrostatic interactions are replaced by an increase in hydrophobic interactions, which may lead to a slight distortion of the coiled coil superstructure. However, the exact structural change resulting in the increase of the ellipticity ratio to ∼1.1 is not fully understood at present. In accordance with the CD data, AUC shows that the relative concentrations of unimers (range: 21-32 wt %), dimers (range: 55-62 wt %), and tetramers (range: 11-17 wt %) for mPEG(750)-EEK are essentially pH-independent (average molecular weight between 6.25 and 6.9 kDa between pH 3.7 and 9.8). For mPEG(2000)-EEK, however, an increase in average molecular weight with decreasing pH is observed (Figure V in the Supporting Information); at pH 3.7, AUC indicates the presence of 35 wt % higher oligomers (around nonamers) in addition to 35 wt % dimers and 30 wt % unimers. In contrast, at pH 7.4 and pH 9.8 dimeric assemblies are predominant, accounting for 80 and 70 wt %, respectively. Self-Organization of X-EEE. Concentrationdependent CD spectra reveal that both Ac-EEE and the peptide segments of the mPEG-conjugates are predominantly (about 90%) in the random coil conformation at pH 7.4 (Figure VI in the Supporting Information). In the random coil conformation, the peptides do not have the ability to organize in coiled coil superstructures and induce self-assembly of the block copolymers. This is supported by AUC experiments at a concentration of 100 µM, which show that Ac-EEE contains ∼95 wt % unimers and mPEG(750)-EEE and mPEG(2000)-EEE contain ∼90 and ∼85 wt % unimers, respectively (Table 2). While the peptide, in addition, contains ∼5 wt % dimeric or larger (i.e., defined, and not formed by unspecific association) aggregates, mPEG(750)-EEE and mPEG(2000)-EEE contain an additional ∼10 and ∼15 wt % dimeric aggregates, respectively. Figure 3 illustrates the pH-dependent coil-to-helix transition which occurs due to neutralization of negative charges on the L-glutamic acid residues at low pH values. The presence of an isodichroic point at 204 nm (Figure VII in the Supporting Information) suggests that this transition occurs via a two-state mechanism. Between pH 3 and 4 the ellipticity ratio ([θ]222/[θ]208) reaches values of about 1.05, indicating that the coil-to-helix transition is accompanied by coiled coil formation (Figure 3b). Complementary AUC experiments, carried out at pH 3.7, indicate that Ac-EEE consists of ∼10 wt % unimeric molecules, ∼34 wt % dimeric aggregates, and ∼56 wt % tetrameric aggregates, while mPEG(750)-EEE consists of ∼28 wt % unimeric molecules and ∼72 wt % tetrameric aggregates and mPEG(2000)-EEE of almost exclusively (∼97 wt %) dimeric aggregates (the other ∼3 wt % are tetrameric aggregates). Below pH 3 all compounds start to precipitate, most probably because of the uncontrolled formation of very high aggregates.
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Figure 3. Plots showing the pH dependence of (a) the helix content and (b) [θ]222/[θ]208 for X-EEE (PBS, 100 µM).
The transition midpoint for Ac-EEE, defined as the pH where 50% helix content is present, occurs at pH ∼4.4, which is the pKa value for the carboxylate side chain group of L-glutamic acid. Interestingly, however, the coil-to-helix transition of the mPEG conjugates occurs at a higher pH, as observed by a shift in transition midpoint from pH ∼4.4 to pH ∼5.2 for mPEG(750)-EEE and to pH ∼5.7 for mPEG(2000)-EEE. This increase in transition midpoint can be explained by the formation of a PEG shell around the peptide. In addition to the stabilizing effect of PEG on peptide secondary structure,8 the protonated form of L-Glu can form hydrogen bonds with PEG which may shift the pKa and also contribute to the observed increase in transition midpoint. Self-Organization of X-KKK. At pH 7.4, CD spectra of both the peptide and diblock copolymers show an increase in helix content with increasing concentration, which suggests the presence of a concentration-dependent aggregation process (Figure 4a and Figure VIII in the Supporting Information). Both mPEG conjugates possess a somewhat higher helix content than the peptide. Figure 4b, however, shows that the ellipticity ratio [θ]222/[θ]208 for both Ac-KKK and the two block copolymers remains below 0.88 over the entire concen-
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Figure 4. Plots showing the concentration dependence of (a) the helix content and (b) [θ]222/[θ]208 for X-KKK in PBS at pH 7.4.
tration range measured, which indicates that the peptide chains predominately remain in their unimeric state at neutral pH. This can be explained by the net positive charge of the peptide block at neutral pH, which prevents aggregation due to interhelical electrostatic repulsions. AUC experiments confirm the unimeric character at pH 7.4 (Table 2). Ac-KKK consists of ∼95 wt % unimers and ∼5 wt % small aggregates, mPEG(750)-KKK consists of 77 wt % unimers and ∼23 wt % higher oligomers, and mPEG(2000)-KKK consists of ∼35 wt % unimers, ∼45 wt % dimeric aggregates, and ∼20 wt % other higher oligomers. Figure 5a shows the pH dependence of the helix content. The increase in helix content together with the observation of an isodichroic point at 204 nm in the CD spectra (Figure IX in the Supporting Information) suggests the presence of a pH-dependent coil-to-helix transition, proceeding via a two-state mechanism. The transition is, however, not as clearly reflected in the CD plots as it was the case with X-EEE. The ellipticity ratio reaches a value of ∼1.0 at pH 10, suggesting the formation of a coiled coil superstructure. As indicated by sedimentation equilibrium analysis at pH 9.8, the amount of unimeric molecules is, however, still large. Ac-KKK consists of ∼55 wt % unimers and ∼45 wt %
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Figure 5. Plots showing the pH dependence of (a) the helix content and (b) [θ]222/[θ]208 for X-KKK (PBS, 100 µM).
dimeric aggregates, and mPEG(2000)-KKK consists of ∼22 wt % unimers and ∼78 wt % dimeric aggregates. The difference in pH sensitivity between the X-EEE and X-KKK analogues is striking. However, it has been suggested that the protonated form of L-Glu (at low pH) enhances coiled coil formation by packing in a better way in the hydrophobic core than deprotonated L-Lys,7b which may be a possible explanation. Self-Organization of Heteromeric Coiled Coils. Interpolyelectrolyte complexes were prepared at pH 7.4 from equimolar mixtures of Ac-EEE and Ac-KKK, mPEG(750)-EEE and mPEG(750)-KKK, and mPEG(2000)-EEE and mPEG(2000)-KKK. In the following these interpolyelectrolyte complexes will be referred to as heteromeric peptide, heteromeric mPEG(750) conjugate, and heteromeric mPEG(2000) conjugate, respectively. In contrast to the corresponding homooligomers, the formation of these heterooligomers is promoted by relief in electrostatic repulsions between equally charged peptide chains and attractive interhelical interactions between oppositely charged peptide chains. CD experiments at different concentrations reveal R-helical CD spectra and a decrease in mean residue ellipticity with increasing concentration (see Figure X in the Supporting Information). Figure 6 shows the concentration dependence of the helix content and [θ]222/[θ]208 ratio
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Figure 6. Plots showing the concentration dependence of (a) the helix content and (b) [θ]222/[θ]208 for an equimolar mixture of X-EEE and X-KKK in PBS at pH 7.4.
determined from the CD spectra at pH 7.4. All complexes show an increase in helix content with concentration, with the diblock copolymers having a significantly higher helix content than the peptide complex. The ellipticity ratios are all relatively constant at 0.96 above 100 µM, which is an indication for the formation of a heteromeric coiled coil at pH 7.4 by association of oppositely charged peptide chains. In contrast, the ellipticity ratios of the individual X-KKK peptides and diblock copolymers at pH 7.4 are significantly smaller and do not suggest the formation of coiled coil superstructures. Furthermore, the individual X-EEE peptides and diblock copolymers are predominately random coils, which is also in contrast to the behavior of the interpolyelectrolyte complexes. Upon dilution below 100 µM, a far-UV shift of the minimum at 208 nm is observed (Figure X in the Supporting Information), which indicates unfolding of the coiled coil aggregates into the individual peptides or block copolymers with a random coil conformation. Sedimentation equilibrium analysis at 100 µM provides further support for the formation of heteromeric coiled coils at pH 7.4 (Table 2). The AUC experiments show that mixing equimolar quantities of anionic (EEE) and cationic (KKK) PEG-peptide block copolymers preferentially results in the formation of tetrameric coiled coils. The relative amount of coiled coil aggregates in the heteromeric mPEG(2000) conjugate (64 wt %) is smaller than that of the mPEG(750) conjugate (89 wt %), which can be explained by the increased steric hindrance of the longer PEG chain on coiled coil formation. As shown in Figure 7a (and Figure XI in the Supporting Information), CD experiments performed at different pH values (at a concentration of 100 µM) reveal only a minimal pH sensitivity of the helix content. Over the investigated pH range, the helix content of the heteromeric mPEG conjugates is much higher than that of the heteromeric peptide: around 60% for the mPEG(750) mixture and 70-80% for the mPEG(2000) mixture, whereas the heteromeric peptide only reaches helix contents between 30 and 40%. The heteromeric mPEG(2000) conjugate is soluble between pH 0 and 12, whereas the heteromeric mPEG(750) conjugate and heteromeric peptide precipitate below pH 3.0 and 4.5, respectively. Analytical ultracentrifugation experiments show no significant pH sensitivity of the heteromeric coiled coil aggregates, as evidenced by plots of the
Figure 7. Plots showing the pH dependence of (a) the helix content and (b) [θ]222/[θ]208 for an equimolar mixture of X-EEE and X-KKK in PBS at pH 7.4 (total peptide concentration: 100 µM).
average molecular weight vs pH for both the peptide and diblock copolymers (see Figure XII in the Supporting Information). Cytotoxicity and Red Blood Cell Lysis. In vitro cytotoxicity was determined using the MTT method and B16 F10 mouse melanoma cells. Neither the peptides nor the PEG-b-peptide diblock copolymers cause significant red blood cell lysis. For all the investigated samples, the hemoglobin release over 1 h is 5.0 0.004 ( 0.0005 >5.0 >5.0 >5.0 1.6 ( 0.02 1.6 ( 0.03 1.6 ( 0.07 0.13 ( 0.01 0.06 ( 0.009 0.16 ( 0.01 2.2 ( 0.09 2.0 ( 0.13 1.7 ( 0.14
which may explain the lower IC50 values as compared with the nonionic dextran. The X-KKK compounds have the lowest IC50 values, probably because of their positive charge, which allows strong interactions with the negatively charged phospholipid cell membranes. The XEEK-based samples and the heterooligomeric polyelectrolyte complexes are less toxic than the X-EEE and X-KKK samples, probably due to the interhelical salt bridges that are involved in coiled coil formation, which would lead to (partial) screening of the positive and negative charges. DNA Complexation. It is well-known that cationic polymers such as PLL and poly(ethylene imine) (PEI) can form interpolyelectrolyte complexes with DNA.24 PLL and PEI have been widely investigated as nonviral vectors for gene delivery. Although safer than viral vectors, their inability to promote efficient transfection and their high nonspecific toxicity remains a problem.25 The cationic mPEG-KKK diblock copolymers described here do not show any red blood cell lysis at pH 7.4 (1 h), and their IC50 values are up to 40 times higher than that of PLL. Because of this improved biocompatibility and their ability to complex DNA, the mPEG-KKK conjugates may be promising nonviral vectors for gene delivery. To assess their potential for DNA complexation, the cationic peptide Ac-KKK and the corresponding mPEG diblock copolymers were mixed with DNA at molar ratios between 50:1 and 1000:1 and subsequently analyzed in a gel retardation assay. Figure 9
(and Figure XIV in the Supporting Information) reveals that, in comparison with the free DNA in lane 1, mixtures of X-KKK and DNA at molar ratios above 100:1 show retardation.26 At identical molar ratios X-KKK:DNA, retardation is most pronounced for the mPEG(2000) conjugate, reflecting the size of the DNA interpolyelectrolyte complex. Formation of DNA complexes does lead not only to retardation but also to a loss in ethidium bromide fluorescence intensity (Figure 9). This arises due to the disruption of the stacked DNA base pairs upon complexation of the polynucleotide backbone with the positively charged peptide segments. Conclusions Biological synthetic hybrid block copolymers are an attractive class of materials due to the potential synergetic interactions between the constituent segments.27 In this paper, we have systematically investigated the effect of manipulation of peptide primary structure on the self-assembly properties of PEG-b-peptide hybrid block copolymers. Using the LAEIEAK-based de novo coiled coil sequence, which was already described in an earlier publication,8 as the starting point, the X-EEK peptide and diblock copolymers are obtained by exchanging amino acid residues in heptad repeat positions a and d, whereas the X-EEE and X-KKK peptide and diblock copolymer analogues involve modification of positions c, e, and g. Despite the differences in peptide primary structure, all peptides and conjugates retain their ability to form coiled coils. According to CD and AUC experiments, the self-organization of the mPEGEEK diblock copolymers can be described as a two-state equilibrium between discrete unimers and dimeric coiled aggregates. The mPEG-EEE and mPEG-KKK diblock copolymers show pH-dependent coil-to-helix transitions and only fold into homomeric coiled coil aggregates at low and high pH, respectively. However, the enhanced pH control over folding and unfolding of the X-EEE and X-KKK materials in comparison with the X-EEK-based samples is accompanied by a slight decrease in the specificity of the oligomerization properties. The ability to control self-assembly properties, such as degree of oligomerization and pH sensitivity, via directed single or 2-fold amino acid substitutions in the peptide primary structure is of considerable significance, since it was found that the biological properties (e.g., cytotoxicity) of the PEGylated coiled coils correlate with their self-assembly behavior. Future work will focus on
Macromolecules, Vol. 38, No. 3, 2005
tailoring these stimuli-responsive diblock copolymers for specific biomedical applications, in particular drug delivery systems. In this respect, we have already shown that the constructs have a favorable biocompatibility profile and that mPEG-KKK conjugates can be used to complex DNA. Acknowledgment. This work was supported by the International Max Planck Research School, the Boehringer Ingelheim Fonds, and the Deutsche Forschungsgemeinschaft (Emmy Noether-Program). The authors are grateful to Prof. H. Ringsdorf and Prof. D. Schubert for their help and discussions and to H. Nuhn for his help with the RP-HPLC experiments.
PEG-Based Hybrid Block Copolymers 769
(8) (9) (10) (11) (12) (13) (14) (15)
Supporting Information Available: Analytical data, CD, and AUC characterization and additional red blood cell lysis and gel retardation assay results. This information is available free of charge via the Internet at http://pubs.acs.org.
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References and Notes
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