Article pubs.acs.org/Macromolecules
Aqueous Self-Assembly of a Protein-Mimetic Ampholytic Block Copolypeptide Jing Sun,*,†,§ Peter Č ernoch,‡,§ Antje Völkel,§ Yuhan Wei,† Janne Ruokolainen,∥ and Helmut Schlaad*,⊥,§ †
School of Polymer Science and Engineering, Qingdao University of Science and Technology, 53 Zhengzhou Road, Qingdao 266042, China ‡ Institute of Macromolecular Chemistry, Heyrovského nám. 2, 162 06 Praha 6, Czech Republic § Department of Colloid Chemistry, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, 14424 Potsdam, Germany ∥ Department of Applied Physics, Aalto University Nanomicroscopy Center (Aalto-NMC), Puumiehenkuja 2, 02150 Espoo, Finland ⊥ Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany S Supporting Information *
ABSTRACT: This report describes the aggregation behavior of an ABC-type ampholytic block copolypeptide, poly(ethylene oxide)-blockpoly(L-lysine)-block-poly(L-glutamate), in aqueous media in dependence of pH. Polypeptide secondary structures and self-assemblies are investigated by circular dichroism (CD), Fourier transform infrared (FT-IR) and NMR spectroscopy, zeta potential measurements, analytical ultracentrifugation (AUC), dynamic/static light scattering (DLS/SLS), and cryogenic transmission electron microscopy (cryoTEM). The polymer chains tend to form vesicles when the hydrophobic polypeptide helix is located at the chain end (acidic pH) and are existing as single chains when it is located in the center and flanked by the two hydrophilic segments (basic pH). Precipitation occurs in the intermediate pH range due to polyion complexation of the charged polypeptide segments.
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INTRODUCTION In the past few years, increasing interest has been given to the self-assembly of block copolymers in aqueous solution because of their potential applications in nanoscience and nanotechnology, such as carriers for drug and gene delivery, diagnostic imaging, and nanoreactors.1−7 Meanwhile, selfassembly is an essential process in biological systems; various kinds of bio(macro)molecules, including proteins, DNA, and phospholipids, can spontaneously assemble into a diverse range of hierarchical structures.8 It is well-known that the secondary structure of proteins is based on the assembly of peptidic segments in α-helices and β-sheets via intra- and intermolecular interactions between the functional groups of residual amino acids. These assemblies play a key role for the biological behavior of proteins. In order to mimic biological activity of natural proteins, chemical and material research has been pushed to incorporate polypeptides into synthetic materials to obtain biohybrid (macro)molecules. These biohybrids (or chimeras)9 are approaching proteins in their complexity, functionality, and performance, thus making them important elements for research and applications in biomimetic materials and the biomedical field.10−15 Many polypeptide-based block copolymers have been synthesized and studied according to their aggregation behavior in dilute solutions.16−31 Most studies deal © XXXX American Chemical Society
with diblock copolymers; however, very few are focused on ABC-type triblock copolymers.24,32 In the present contribution, we describe the pH-dependent aggregation behavior of an ampholytic ABC block copolypeptide, i.e., poly(ethylene oxide)-block-poly(L-lysine)-block-poly(Lglutamate) (PEO-PLLys-PLGlu; low pH: coil−coil−rod/high pH: coil−rod−coil), in dilute aqueous solution, which is distinctively different from that of the reported “schizophrenic” PLLys15-PLGlu15 diblock oligomer19 or PLLys40-(PLGlu15)4 linear-dendron-like polyampholyte31 (coil−rod/rod−coil) or even the poly(N-isopropylacrylamide)-block-poly((L-lysine)-co(L-glutamate))26 (coil−rod/coil−rod). The two polypeptide segments exhibit complementary solution and conformational behavior in dependence of pH; i.e., poly(L-lysine) is a soluble random coil in acidic media but insoluble α-helix in basic media, which is reversed for the poly(L-glutamate). The polypeptide secondary structure and the aggregation behavior in aqueous media are examined by NMR, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, zeta potential, analytical ultracentrifugation (AUC), dynamic/static Received: April 19, 2016 Revised: July 4, 2016
A
DOI: 10.1021/acs.macromol.6b00817 Macromolecules XXXX, XXX, XXX−XXX
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Malvern Zetasizer Nano ZS. Zeta-potentials (ζ) were calculated with the Smoluchowski’s formula: ζ = μEη/ϵ, where η denotes the viscosity and ϵ the permittivity of the solution. Solutions were prepared by dissolving the polymer in water (2 mg mL−1; pH ∼ 1.9 or 13.0) followed by titration of the initial solution to neutral region using 1 M HCl or 1 M NaOH, respectively. Analytical Ultracentrifugation. AUC measurements were performed with the Rayleigh interference detection system of a Beckman Coulter XLI analytical ultracentrifuge. Depending on the sedimentation behavior of the sedimenting species rotational speeds of 20 000, 40 000, and 60 000 rpm for velocity runs have been chosen. Sedimentation coefficient distributions have been calculated using the software package SEDFIT (version 12.52 beta P. Schuck 2011).33 Equilibrium experiment data have been collected in a speed range of 25 000−45 000 rpm. Data were evaluated with the program MSTAR (Kristian Schilling, Nanolytics, Germany).34 Density measurements were carried out on a density meter DMA5000 (Anton Paar, Graz, Austria). Dynamic/Static Light Scattering. DLS/SLS measurements were performed using ALV-7004 multiple tau digital correlator equipped with CGS-3 compact goniometer system, 22 mW He−Ne laser (wavelength λ = 632.8 nm), and a pair of avalanche photodiodes operated in a pseudo-cross-correlation mode. Dynamic depolarized light scattering (DDLS) was measured on the ALV setup using a Glan−Thompson prism in the front of the optical detector as analyzer. In all DLS experiments, the measured intensity correlation functions g2(t) were analyzed using the algorithm REPES35 performing the inverse Laplace transformation according to
light scattering (DLS/SLS) measurements, and cryogenic transmission electron microscopy (cryo-TEM).
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EXPERIMENTAL PART
Materials. The N-carboxyanhydrides (NCAs) of Nε-benzyloxycarbonyl-L-lysine (ZLLys; Novabiochem) and γ-benzyl-L-glutamate (BLGlu; Novabiochem) were synthesized from the corresponding amino acid and bis(trichloromethyl)carbonate (triphosgene; SigmaAldrich) (1.5 equiv) in dry tetrahydrofuran (THF; Sigma-Aldrich) solution at 50 °C. After 1.5 h, the mixtures were precipitated by an excess of heptane, and crude products were recrystallized three times from ethyl acetate/heptane; yield: 75−82%. α-Methoxy-ω-amino-poly(ethylene oxide) (PEO-NH2) was purchased from Rapp Polymere (Tübingen, Germany). The numberaverage molar mass (Mn) of the PEO sample was 1.9 kDa, dispersity (Đ) = 1.04, as determined by size exclusion chromatography (PEO calibration). Polymer Synthesis. The ring-opening polymerization (ROP) of ZLLys NCA (14.5 g) was carried out in dry N,N-dimethylformamide (DMF; Sigma-Aldrich) solution (140 mL) using PEO-NH2 (1.5 g) as the initiator ([ZLLys NCA]0/[NH2]0 = 60). The reaction mixture was stirred for 5 days at room temperature under an argon atmosphere. After consumption of the first monomer, BLGu NCA (12.4 g, [BLGlu NCA]0/[NH2]0 = 60) was added, and the mixture was stirred for another 5 days at room temperature. The PEO−PZLLys−PBLGLu triblock copolymer product was precipitated into large amount of diethyl ether and dried under vacuum at room temperature for 2 days; gravimetric yield: 21.2 g (87%). 1H NMR (400.1 MHz, DMSO-d6): δ/ ppm = 1.27−1.87 (b, (CH2)3(−CH)), 2.0, 2.2 (b, (CH2)2(−CH)), 2.95 (m, CH2(−NH)), 3.50 (b, CH2CH2O), 3.82 (m, CH, Lys), 3.91 (m, CH, Glu), 5.02 (m, CH2(−Ph)), 7.25 (m, Ph), 8.21 (b, NH); Mn = 30.6 kg mol−1, calculated on the basis of peak integrals at 3.50 ppm (PEO, 4 × 42 H), 3.82 (PZLLys), and 3.91 (PBLGlu). SEC (eluent: Nmethyl-2-pyrrolidone, NMP + 0.5 wt % LiBr, 70 °C, stationary phase: PSS-GRAM, detector: RI, calibration: polystyrene): Đ = 1.07. PEO−PZLLys−PBLGLu (1 g) was deprotected by reacting with 33% HBr−glacial acid (4 equiv with respect to ZLLys and BLGlu repeat units) at 0 °C for 2 h. The PEO−PLLys−PLGlu was precipitated with an excess of diethyl ether to get a white solid and was dried under vacuum at room temperature for 2 days; gravimetric yield: 0.5 g (89%). 1H NMR (400.1 MHz, TFA-d): δ/ppm = 0.95− 1.50 (b, (CH2)3(−CH)), 1.9, 2.3 (b, (CH2)2(−CH)), 2.88 (m, CH2(−NH)), 3.58 (b, CH2CH2O), 4.25 (m, CH, Lys), 4.48 (m, CH, Glu). Preparation of Polymer Solutions. 0.5 g of polymer was directly dissolved in ∼200 mL of deionized water (pH < 4) in an ultrasonic bath to give a colloidal solution, which was then dialyzed for 1 week against deionized water and freeze-dried. The lyophilized powder was directly dissolved into solutions with desired pH value, which was adjusted by HCl or NaOH solutions. After stirring overnight, exact pH values of the solutions were measured using a calibrated pH electrode. Suspensions of polymer (∼0.15 wt %) in 0.5 M NaCl solution were titrated to the desired pH value using 1 M NaOH or 1 M HCl in saline solutions. The titrated solutions were stirred overnight. If required, a series of different concentrations were prepared by dilution of this stock solution with 0.5 M NaCl and readjustment of the pH value. Spectroscopy. 1H NMR measurements were carried out at room temperature using a Bruker DPX-400 spectrometer operating at 400.1 MHz. DMSO-d6, D2O (Aldrich), DCl (20% solution in D2O, AppliChem GmbH), and NaOD (30% solution in D2O, Carl Roth GmbH & Co. KG) were used as solvents; signals were referenced to the signal of residual protonated solvent at δ = 4.79 ppm. Circular dichroism (CD) spectra were obtained with a JASCO J 715 spectrometer using a quartz cuvette with 1 mm path length. For each measurement ten accumulations were averaged. FT-IR spectra were recorded on a BioRad 6000 FT-IR; samples were measured in the solid state using a single reflection diamond ATR. Zeta-Potential Measurements. The electrophoretic mobility (μE) of the aggregates in dependence of pH was measured on a
∫
g 2(t ) = 1 + β[ A(t ) exp(− t /τ ) dτ ]2 n
= 1 + β[∑ Ai exp(− t /τi)]2 i=1
(where t is the delay time of the correlation function and β an instrumental parameter) and yielding distribution A(τ) of relaxation times τ. The relaxation time τ is related to the diffusion coefficient D and relaxation (decay) rate Γ by the relation Γ = 1/τ = Dq2, where q is the scattering vector defined as q = (4πn/λ) sin(θ/2) where n is the refractive index of the solvent and θ is the scattering angle. The hydrodynamic radius Rh of the particles can be calculated from the diffusion coefficient using the Stokes−Einstein equation D = kBT/ 6πηRh, where T is absolute temperature, η the viscosity of the solvent, and kB the Boltzmann constant. SLS experiments were carried out at scattering angles from 40° to 150° with 10° steps. The data were evaluated by a standard calculation package ALV-Stat, provided standard double extrapolation Zimm analysis and yielding the apparent molar mass of aggregates (Mwapp), radius of gyration (Rg), second virial coefficient (A2), and hydrodynamic radius R0h obtained by extrapolation of measured data to q2 = 0 and concentration c = 0 (dynamic Zimm plot). Refractive index increments dn/dc of the polymer solutions were measured with the interferometric refractometer ScanRef (NFT Nanofilm Technologie GmbH, Göttingen, Germany); dn/dc (λ = 633 nm) = 0.1911 mL g−1 (pH 2.0) and 0.2030 mL g−1 (pH 11.7) (water), 0.0689 mL g−1 (pH 2.0) and 0.0568 mL g−1 (pH 12.3) (0.5 M NaCl). For DLS/SLS analysis, a series of stock solutions with different pH and concentration values were prepared. Samples with a lower concentration were made by consequent diluting of the stock solution with water with preadjusted pH (same as pH of stock solution) or with saline solution and further readjustment of pH. Samples were filtered through 0.45 μm PVDF filters directly before measurement and flamesealed into a glass ampule. Cryogenic Transmission Electron Microscopy. The vitrified specimens were prepared using a Vitrobot (FEI, Inc.). Grids with lacey carbon films were glow discharged to produce a hydrophilic surface, and a 5 μL droplet of the aqueous nanotube suspension was deposited on the surface. The droplet was blotted by filter paper for 1−2 s followed by 1−2 s draining and plunged into liquid ethane to obtain a vitrified thin film. The vitrified specimens were examined using a FEI B
DOI: 10.1021/acs.macromol.6b00817 Macromolecules XXXX, XXX, XXX−XXX
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signal is detected at λ < 200 nm, however, indicating the coexistence of α-helices (strong positive signal) and random coils (strong negative signal).36 It is known that PLGlu (pKa ∼ 4.3) adopts a noncharged α-helical conformation at low pH and a charged random coil conformation at high pH.17,19 Contrary to this, PLLys (pKa ∼ 10.0) forms a random coil at low pH and turns into an α-helix at high pH.22 Thus, at either low or high pH, there should be half proportion of polypeptide segment adopting random coil conformation and half adopting α-helical structure. At pH 4.1 and 11.7 (coagulation occurs for solutions in the intermediate pH range of ∼4.5−10.5; see below), there is evidence for the existence of β-sheet as indicated by the negative CD peak absorption with single minimum at ∼218 nm. This observation is consistent with previous studies, showing that PLLys/PLGlu polyion complexes (multilayers) form β-sheets despite the fact that the single polypeptide chains adopt random coil conformations at neutral pH.37 It is further noteworthy that the secondary structure formation of PLLys at very high pH may be affected by the way of sample preparation, especially whether or not the polymer was subjected to freeze-drying (lyophilization).38 PLLys seemed to form a β-sheet (CD data not shown) when the freeze-dried polymer powder was directly dissolved in water at pH 13, as described in the Experimental Part. However, the α-helix developed (Figure 2) in the dialyzed polymer solution adjusted to pH 13. Freeze-dried polymer solutions were further studied by FTIR spectroscopy for analysis of the secondary structure of polymer chains at different pH (Supporting Information). For the sample prepared at pH 2.5, the absorption peaks at ṽ ∼ 1648 cm−1 (CO stretching vibration, amide I) and 1542 cm−1 (N−H bending vibration, amide II) indicate the presence of an α-helical structure.39 The random coil secondary structure could be recognized by a typical absorption peak at ∼1650 cm−1.40 With increasing pH, the amide I absorption band at 1648 cm−1 shifts gradually to 1618 cm−1, which is characteristic of the β-sheet conformation. Moreover, the peak at 1542 cm−1 broadens and shifts to 1527 cm−1 at pH 7, which further verifies that the polymer exists in β-sheet form. In alkaline solution at pH 12.6, the amide I/II bands are shifted back to 1648 and 1550 cm−1, respectively, which can again be attributed to random coil/α-helix conformations. In summary, the CD and FT-IR spectroscopic data indicate that the secondary structure of the ampholytic block copolypeptide goes through a change from random coil/αhelix (low pH) to β-sheet (intermediate pH) to α-helix/ random coil (high pH) (see Figure 1b). Solution Behavior: Zeta Potential and NMR Spectroscopy. The 0.2 wt % polymer solution was examined according to the zeta potential (ζ) as a function of pH (Figure 3). The zeta potential is positive, i.e., +35 mV, at pH 2.3 and is gradually decreasing with increasing pH, reaching a minimum value of −33 mV at pH 12.4. Considering the pKa values of PLLys (∼10.0) and PLGlu (∼4.3) (see above), the positive ζ values at low pH indicate the presence of cationic ammonium groups from PLLys; the PLGlu carboxylic acid groups are fully protonated thus noncharged at pH 2.4. With increasing pH, the degree of ionization of PLLys gradually decreases and that of PLGlu increases, finally leading to negative ζ values due to the presence of anionic carboxylates at high pH. The similar zeta-potential absolute values at high/low pH and occurrence of the isoelectric point (net zero charge) at pH 8 can be well
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RESULTS AND DISCUSSION The PEO−PZLLys−PBLGlu triblock copolymer, as prepared by PEO-NH2 initiated ROP of protected amino acid NCA, has an absolute number-average molar mass of Mn = 30.6 kg mol−1 (1H NMR) and narrow apparent molar mass distribution, dispersity Đ = 1.07 (SEC) (Mw = MnĐ = 32.7 kg mol−1). The molar masses of three block segments are 1.9, 16.0, and 12.7 kDa, respectively, corresponding to 42 (PEO), 61 (ZLLys), and 62 (BLGlu) repeat units. Subsequent hydrolysis of protecting groups was quantitative, as confirmed by 1H NMR analysis (data not shown). The chemical structure of the PEO42− PLLys61−PLGlu62 is displayed in Figure 1a.
Figure 1. (a) Chemical structure and (b) pH-dependent conformation (secondary structure) of PEO42−PLLys61−PLGlu62.
Secondary Structure Analysis: CD and FT-IR Spectroscopy. The secondary structure of PEO42−PLLys61− PLGlu62 at 0.2 wt % in water in a pH range of 1.9−13.0 was first examined with CD spectroscopy; spectra are shown in Figure 2. The spectra obtained at pH 1.9−3.1 and 12.3−13.0 show two negative bands at λ ∼ 208−212 and 221−222 nm, which are attributable to the presence of α-helices. No CD
Figure 2. CD spectra of 0.2 wt % solutions of PEO42−PLLys61− PLGlu62 in water at pH 1.9−4.1 (left) and pH 11.7−13.0 (right). C
DOI: 10.1021/acs.macromol.6b00817 Macromolecules XXXX, XXX, XXX−XXX
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pH 3.4, the zeta potential was found to be +32.5 mV. (Note: the measured ζ values might be slightly affected by the creation of NaCl (screening of charges) or by small variations of solution pH.) The results of 1H NMR analysis of the triblock copolymer in strongly acidic (0.9 M DCl in D2O) and basic (1.0 M NaOD in D2O) media add to the zeta-potential measurements. The spectra only reveal the proton signals of the two hydrophilic blocks and lack those of the hydrophobic polypeptide block, which is PLGlu at low pH and PLLys and high pH (Supporting Information). Solution and Aggregation Behavior: Analytical Ultracentrifugation and Light Scattering. As mentioned above, PEO42−PLLys61−PLGlu62 could be well dispersed in water at acidic pH and basic pH but not in the intermediate pH range of ∼4.5−10.5 (as the PEO block is seemingly too short to stabilize the hydrophobic polypeptide segments). We first studied 0.17− 0.19 wt % aqueous polymer solutions at pH 2.0 and 12.3 in the absence of additional salt. Results from dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) measurements are shown in Figure 4. DLS reveals one (pH 2.0) or two (or more) (pH 12.3) diffusive dynamic modes, which are commonly observed in dilute polyelectrolyte systems in the absence or presence of little amounts of salt.41−44 The slower mode, making on average 95+% of the total scattered intensity, may be assigned to various types of aggregates formed in polymeric systems. The faster mode is usually reflecting mutual cooperative diffusion of polyions and counterions or can be assigned to molecularly dissolved polymer chains. AUC sedimentation coefficient distributions, g*(s), on the other hand, indicate the presence of two species sedimenting at different speeds. The slower sedimenting fraction at s ∼ 1 Sv (1 Svedberg = 10−13 s) can be assigned to individual polymer chains, by molar mass determination through AUC equilibrium measurements, and makes about 91 wt % (pH 2.0) to 78 wt % (pH 12.3) of the sample (as determined by refractive index difference in the interference optics). The faster sedimenting fractions at s ∼ 200 Sv (pH 2.0) and 10 Sv (pH 12.3) are considered to be aggregates of yet unknown structure (DLS/ SLS results are inconclusive, data not shown). It is evident that, however, the amphiphilic PEO42−PLLys61−PLGlu62 dissolves as
Figure 3. Zeta potential of 0.2 wt % solutions of PEO42−PLLys61− PLGlu62 in water at pH 2.3−12.4.
explained by the equal lengths of polypeptide blocks in the copolymer. The values of the zeta potential indicate which of the peptide segments is charged (cationic PLLys at low pH and of anionic PLGlu at high pH) and contributes to the stabilization of the chains or particles in solution. The PEO segment remains hydrophilic and costabilizes the particles in the whole pH range. However, at intermediate pH when PLLys and PLGlu form an insoluble polyion complex and precipitation occurs, the PEO component is not sufficient to stabilize the particles to prevent coagulation. The pH-induced transition between cationic and anionic species is reversible within a time frame of a few hours. To a 0.2 wt % polymer solution at pH 11.7, exhibiting a ζ potential of −33.0 mV, was added 1 M aqueous HCl to decrease the pH to 3.1. After 3 h the zeta potential was measured to be +34.2 mV (a lower ζ value was measured after 1 h indicating nonequilibrium state). The pH of the solution was then again adjusted to pH 11.7 by the addition of 1 M NaOH, and the zeta potential was measured 3 h later to give a value of −31.5 mV, i.e., close to the original value. After addition of HCl to reach
Figure 4. (a) Apparent particle size distributions (DLS, 90°) and (b) sedimentation coefficient distributions (AUC, 40K/60K rpm) of (freshly prepared) ∼0.2 wt % solutions of PEO42−PLLys61−PLGlu62 in water at pH 2.0 (dashed lines) and 12.3 (solid lines). D
DOI: 10.1021/acs.macromol.6b00817 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules single chain with marginal tendency toward aggregation (otherwise as suggested by DLS) at ∼0.2 wt % in water. This implies that the hydrophobic polypeptide segment (PLGlu outer block at low pH and PLLys middle block at high pH) can be shielded from the water phase by the two other hydrophilic blocks to prevent aggregation. This wrapping of the hydrophobic α-helical block with the hydrophilic segments could be compared, albeit on a much less sophisticated level, with the folding of protein chains into a tertiary structure. The aggregation behavior of PEO42−PLLys61−PLGlu62 at ∼0.1 wt % was considerably affected by the addition of salt, i.e., 0.5 M NaCl, due to a screening of charges. At acidic pH 1.3− 2.6, 70−80 wt % of the polymer chains are incorporated into aggregates while 20−30 wt % remain as single chains, see Figure 5 (the composition of solutions were determined from
pared solutions; same solutions were analyzed ∼3 months later by AUC); results are summarized in Table 1, and exemplary DLS and SLS data obtained at pH 2.0 are shown in Figure 6
Figure 5. Weight fractions of sedimenting species (single chains: s ∼ 1 Sv; aggregates: s > 10 Sv) in ∼0.1 wt % solutions of PEO42−PLLys61− PLGlu62 in 0.5 M aqueous NaCl at pH 1.3−2.6 (dashed lines) and pH 11.0−12.4 (solid lines). (Polymer solutions were analyzed ∼3 months after their preparation.)
Figure 6. Concentration- and angle-dependent (a) DLS and (b) SLS data for 0.04−0.14 wt % of (freshly prepared) PEO42−PLLys61− PLGlu62 in 0.5 M aqueous NaCl at pH 2.0 at room temperature.
(all DLS/SLS data are collected in the Supporting Information). The aggregates appear to have a spherical shape, as confirmed by DDLS (see Supporting Information) and cryoTEM (Figure 7), more precisely should be unilamellar vesicles, as indicated by the ratios Rg/R0h close to unity (0.87 and 0.98− 1.07),25,45 measuring about 100−170 nm in diameter (Table 1). Note that Rg/R0h = 1 is the theoretical value for a uniform hollow sphere with an infinitely thin wall; deviations may originate from shape fluctuations and variations in size.22 Vesicle formation may be expected considering the composition of the chains (hydrophilic weight fraction: ∼0.55) and the intrinsic stiffness of the core-forming PLGlu helix block.46 The value of the second virial coefficient is A2 ≈ 0, within experimental error, and indicates the absence of long-range
refractive index difference measured in AUC sedimentation− velocity experiments). At high pH 11.0−12.4, the situation is reversed with 80−90 wt % single chains and just 10−20 wt % of chains forming aggregates. Seemingly, aggregation of the amphiphilic polymer chains is less pronounced when the two stabilizing hydrophilic segments are the outer blocks (Figure 1b, bottom) and can wrap around the hydrophobic helix to shield it from the water phase. The formation of aggregates is evident when the polypeptide helix is located at the chain end. The aggregates of PEO42−PLLys61−PLGlu62 in 0.5 M NaCl at acidic pH 2.0−3.4 were further analyzed by concentrationand angle-dependent DLS/SLS measurements (freshly pre-
Table 1. Results of Concentration- and Angle-Dependent DLS/SLS Analysis of (Freshly Prepared) 0.04−0.15 wt % PEO42-bPLLys61-b-PLGlu62 in 0.5 M Aqueous NaCl Solutions at pH 2.0−4.1 and Room Temperaturea pH
R0h (nm)
Rg (nm)
2.0 2.2 2.6 3.4 4.1
± ± ± ± ±
± ± ± ± ±
82.5 54.1 60.7 71.4 296
0.7 0.6 0.4 1.1 26
84.3 52.8 52.7 76.3 340
2.3 0.7 2.2 2.8 23
Rg/R0h 1.02 0.98 0.87 1.07 1.15
Mwapp (kg mol−1) (1.44 (5.96 (8.12 (1.29 (5.05
± ± ± ± ±
0.03) 0.06) 0.10) 0.03) 1.30)
× × × × ×
A2 (mol dm3 g−2)
Z 5
10 104 104 105 106
7580 3140 4270 6790 265800
± ± ± ± ±
150 35 55 160 69100
(2.70 (4.81 (5.36 (3.12 (2.86
± ± ± ± ±
0.49) 1.81) 0.43) 1.72) 3.72)
× × × × ×
10−10 10−10 10−10 10−10 10−10
= hydrodynamic radius, Rg = radius of gyration, Mwapp = apparent molar mass, Z = Mwapp/Mwpolymer (= 19 kg mol−1) = apparent aggregation number, and A2 = second viral coefficient. a 0 Rh
E
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are also vesicles, which is supported by their slow sedimentation in AUC (s ∼ 10 Sv, Figure 5).53 Aggregate Morphology: Cryogenic Transmission Electron Microscopy. The aggregates of PEO42−PLLys61− PLGlu62 at ∼0.1 wt % in 0.5 M NaCl solution at pH 2.0 and 12.3 were also subjected to cryo-TEM analysis. For the first attempt, these polymer solutions were used which had already been analyzed by AUC and DLS/SLS; the time period between sample preparation and analysis was on the order of several months. Rather than the expected vesicles (see above) we observed rodlike or fibrous structures as shown in the Supporting Information (Figure S14) (some nonspherical aggregates were also evidenced by AUC (Figure S15)). However, for the freshly prepared polymer solutions, we could observe the unilamellar vesicles with an average diameter of ∼100 nm at pH 2.2 (Figure 7a), whereas no (or barely no) aggregates could be detected at pH 11.8 (Figure 7b), which is in line with previous AUC results (Figure 5) (aggregates are too few in number and/or too large in size to be imaged by cryo-TEM). The thickness of the vesicle wall can be roughly estimated from the dimension of the dark ring seen in Figure 7a to be approximately 5−10 nm (hydrophobic PLGu + protonated PLLys; PEO not seen due to low contrast). pH-Dependent Aggregation Behavior: Summary. The pH-dependent solution and aggregation behavior of PEO42− PLLys61−PLGlu62 in saline solution is summarized in Scheme 1. The polymer chains assemble into vesicles (20−30 wt % remaining as single chains) at low pH and disassemble into single chains (10−20 wt % forming aggregates, presumably vesicles) at high pH; this behavior is different than that of earlier reported PLLys−PLGlu diblock copolypeptides which form large aggregates (wormlike micelles, vesicles, and more complex morphologies) at any pH.19,31 Although precipitation occurs at intermediate pH 4.5−10.5, the transition appears to be reversible as suggested by the zeta-potential measurements. The formation of vesicles is preferred when the polypeptide helix is located at the chain end whereas single chains are predominant when the helix is the middle block and is flanked by two hydrophilic blocks. Rodlike or fibrous structures, however, evolve upon aging of the solutions (over several weeks to months).
Figure 7. Cryo-TEM micrographs of (freshly prepared) 0.1 wt % PEO42−PLLys61−PLGlu62 in 0.5 M aqueous NaCl at (a) pH 2.2 and (b) pH 11.8.
interactions between vesicles in the investigated concentration range. It should be noted that AUC reveals two aggregate species (one sedimenting at s ∼ 20−100 Sv and the other at s > 100 Sv; Figure 5), which cannot be distinguished in the light scattering analyses. Apparent aggregation numbers, i.e., numbers of polymer chains building a vesicle, were calculated from the respective molar masses to be Z ∼ 3140−7580 (Table 1).39 The vesicles formed at pH 2.0−3.4 exhibit interface areas of A = 8πRh2 ∼ 171 000 (±1.6%), 73 600 (±2.3%), 92 600 (±1.3%), and 128 100 nm2 (±3.0%), respectively (= inside and outside surface area of a hollow sphere with radius R). Hence, a single polymer chain ought to cover an average surface area of b2 ∼ 21 nm2 (18.7−23.4 nm2), corresponding to an interchain distance b ∼ 4.6 nm, which is by far larger than the expected helix-tohelix distance and the distance between chains at the core− corona interface of amphiphilic coil−coil diblock copolymer micelles47,48 or vesicles (
