Folding Polymers with Pendant Hydrogen ... - ACS Publications

Yongsheng Gao , Dezhong Zhou , Tianyu Zhao , Xuan Wei , Sean McMahon , Jonathan O'Keeffe Ahern , Wei Wang , Udo Greiser , Brian J. Rodriguez , and ...
15 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Folding Polymers with Pendant Hydrogen Bonding Motifs in Water: The Effect of Polymer Length and Concentration on the Shape and Size of Single-Chain Polymeric Nanoparticles Patrick J. M. Stals,§,† Martijn A. J. Gillissen,§,† Tim F. E. Paffen,† Tom F. A. de Greef,† Peter Lindner,‡ E. W. Meijer,† Anja R. A. Palmans,*,† and Ilja K. Voets*,† †

Institute for Complex Molecular Systems, Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands ‡ Institut Max von Laue-Paul Langevin, F-38042 Grenoble Cedex 9, France S Supporting Information *

ABSTRACT: A set of random copolymers based on a benzene-1,3,5-tricarboxamide functional methacrylate (BTAMA) and oligoethylene glycol methacrylate (oEGMA) with different degrees of polymerization (DP, from 110 to 450) and a 10 mol % loading of BTAMA were prepared using RAFT polymerizations. The pendant BTA units encode for the formation of helical aggregates via 3-fold hydrogen bonding while the oligoethylene glycol side chains provide solubility in water. The copolymers were characterized with size exclusion chromatography, 1H NMR spectroscopy, circular dichroism spectroscopy, light scattering, and small angle neutron and Xray scattering. In dilute aqueous solutions, the copolymers fold intramolecularly and form single chain polymeric nanoparticles (SCPNs) in water. Variable temperature CD spectroscopy showed that the cooling curves are independent of the chain length, indicating a lack of cooperativity in the folding of these copolymers. Scattering studies revealed that the SCPNs have an asymmetric shape. An increase in DP results in an increase of the aspect ratio, while the cross-sectional diameter remains the same at around 3 nm. The elongated shape of the SCPN is proposed to account for the noncooperative folding observed using CD spectroscopy, as such a shape results in a constant local BTA concentration as the copolymer increases in length.

■

uril,17 thymine−diaminopyridine,18 six-point cyanuric acid− Hamilton wedge interactions,19 and hydrophobic interactions.20 BTAs are particularly interesting as supramolecular crosslinking units to produce dynamic SCPNs since they are easy to synthesize and straightforward to incorporate into random copolymers.13,15 Most importantly, BTAs self-assemble into helical aggregates as a result of strong, 3-fold hydrogen bonding between the amides of adjacent BTAs.21 Thermodynamic studies have shown that intermolecular BTA aggregation occurs via a cooperative mechanism in both apolar and polar solutions;21 provided that in the latter case the H-bonds are sufficiently shielded from the solvent.21e,f Introducing a stereogenic center in the side-chains of BTAs induces a preferred helicity in the aggregate.21 Previous studies revealed that the behavior of BTA containing dynamic SCPNs resembles in several ways that of proteins.13a−c For example, BTA-based SCPNs adopt a folded conformation at low temperature while they are unfolded or denatured at high temperatures or after

INTRODUCTION

The topic of single-chain polymeric nanoparticles (SCPNs) attracts increasing attention in the polymer science community, since SCPNs allow for the facile construction of structured nano-objects.1 SCPNs are prepared from polymers possessing pendant groups that can be triggered to induce an intramolecular collapse or folding of the polymer.1 Two categories of single-chain polymeric nanoparticles can be distinguished: nondynamic SCPNs in which the intramolecular cross-links are formed by covalent bonds and dynamic SCPNs in which the cross-links consist of noncovalent or dynamic covalent bonds. In nondynamic SCPNs, amine−isocyanate chemistry,2 Bergman-cyclization,3 dimerization of benzocyclobutenes,4 of coumarines,5 or of benzothiophene dioxide,6 intramolecular azide−alkyne 1,3-dipolar cycloaddition,7 Glaser−Hay coupling,8 and other intramolecular reactions9 have been utilized for intramolecular cross-linking of the polymers. In dynamic SCPNs, disulfide bridges10 or acyl hydrazone groups11 have been explored as well as diamides,12 benzene-1,3,5-tricarboxamides (BTAs),13 2-ureidopyrimidinones (UPys),14 a combination of UPys and BTAs,15 BTA−bipyridines,16 cucurbit[n]© 2014 American Chemical Society

Received: February 5, 2014 Revised: April 8, 2014 Published: April 23, 2014 2947

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

Scheme 1. Schematic Representation of the Folding of BTA Containing Water-Soluble Copolymers into Single-Chain Polymeric Nanoparticles

Table 1. Results of the Copolymerizations of BTAMA and oEGMA Using RAFTa polymer

feed ratio (mA/mB)b

target DP (−)

t (h)c

convn (%)

obsd ratio (mA/mB)b

Mnd (kDa)

Đd (−)

DPth (−)

Mn,the (kDa)

BTAf (no.)

Rh g (nm)

P1 P2 P3 P4

10/90 10/90 10/90 10/90

224 246 399 599

18 23 46 46

n.d. 57 81 77

8/92 9/92 10/90 10/90

25.8 32.2 53.1 55.2

1.24 1.23 1.72 1.86

110h 140 323 461

n.d. 69.8 161.9 230.9

9h 11 32 46

6.1 8.5 13.5 28.9

n.d. = not determined. bMolar ratio of BTAMA/oEGMA. cReaction time in hours. dDetermined by SEC in DMF + 10 mM LiBr, 1 mL min−1, relative to poly(ethylene glycol) standards. eOn the basis of conversion by 1H NMR. fAverage number of BTA units per polymer chain. g Hydrodynamic radius as determined by DLS in water. hEstimated based on reaction time. a

addition of a denaturing agent.13d Furthermore, the introduction of catalytically active centers into the hydrophobic interior of water-soluble BTA-containing SCPNs resulted in efficient catalysis in water.13a−c There are, however, notable differences between proteins and SCPNs. For example, SCPNs are based on disperse polymers and lack the precisely defined sequence of monomers present in biomacromolecules. In addition, many proteins show a strongly cooperative transition from the folded state into an unfolded (coil) state,22 a feature that they share with synthetic mimics such as foldamers23 and polyisocyanides.24 We here continue our investigations on the folding mechanism of water-soluble copolymers with pendant BTA units and how this affects the global conformation of the SCPN. The latter is important in view of applications envisioned in catalysis and sensing, wherein the presence of a compartmentalized structure is important for efficient functioning of the SCPN. In a recent study, we showed that at ambient temperatures the formation of the BTA-based helical aggregate occurs intramolecularly in water and results in an elongated shape of the copolymer (Scheme 1).13d At high temperatures, pendant BTAs disassemble and the copolymer adopts a more globular conformation. The nature of the folding process, however, remained unclear. Classical helix−coil theories such as the Zimm−Bragg25 and Lifson−Roig26 models indicate that the cooperativity of the folding process can be assessed by studying the folding process as a function of the chain length. In the case that the folding process is noncooperativeindicative of the absence of non-neighboring interactions between the pendent BTA unitsthe fraction of helical bonds becomes independent of the chain length.27 We therefore prepared a set of random,

amphiphilic copolymers with a constant ratio between BTA units and water-soluble units but differing in degree of polymerization. A combination of spectroscopic and scattering techniques reveals that the longer polymers behave as small polymers linked together, with patches of BTA units that fold independently.

■

EXPERIMENTAL SECTION

Instrumentation, Materials, and Methods. All commercial reagents and solvents were obtained from Acros, Biosolve, or SigmaAldrich, except for deuterated chloroform, 2-propanol, and water which were purchased from Cambridge Isotopes Laboratories. Oligo(ethylene)glycol methacrylate (oEGMA, Mn = 475 g/mol) was passed through a short column filled with inhibitor remover (Aldrich) before use. AIBN was recrystallized from methanol. All other commerical reagents and solvents were used without any additional purification. 4-Cyano-4-methyl-5-(phenylthio)-5-thioxopentanoic acid was kindly provided by SyMO-Chem (Eindhoven, The Netherlands). BTAMA was prepared according to a previously described procedure.13a NMR spectra were measured on a Varian Mercury Vx 400 MHz and/or a Varian 400MR 400 MHz (400 MHz for 1H NMR and 100 MHz for 13C NMR). Deuterated solvents used are indicated in each case. 1H chemical shifts are reported in ppm downfield from tetramethylsilane (TMS). UV/vis and circular dichroism measurements were performed on a Jasco J-815 spectropolarimeter where the sensitivity, time constant and scan rate were chosen appropriately. The molar circular dichroism Δε was calculated as follows Δε = ((CDeffect)/(32890cl)) wherein c is the concentration in mol L−1 and l is the optical path length in cm. Corresponding temperature-dependent measurements were performed with a PFD-425S/15 Peltier-type temperature controller with a temperature range of 263−383 K and adjustable temperature slope. In all experiments the linear dichroism was also measured and in all cases no linear dichroism was observed. Cells with an optical path length of 0.5 cm were used. Dialysis was 2948

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

Scheme 2. Synthesis of P1−P4

1 H NMR. The polymer was purified by dialysis in THF (for P3 and P4) or by redissolving the polymer in CHCl3/MeOH (1 mL/0.2 mL) and precipitation in pentane four times (for P1 and P2). The polymer was dried under high vacuum at room temperature to a constant weight to obtain a pink sticky material. The spectroscopic data obtained was identical for all polymers. Mn and Đ were determined by DMF-SEC relative to PEO-standards. 1H NMR (CDCl3) for P1−P4: δ = 8.44 (s, Ar−H), 7.37 (s, broad, N−H), 7.19 (s, broad, N−H), 4.08 (s, broad, CO2−CH2), 3.65 (s, broad, CH2−O−CH2), 3.55 (s, broad, CO2−CH2−CH2−O), 3.46 (s, NH−CH2), 3.36 (bs, O−CH3), 2.14− 0.67 (m, CH, CH2, CH3, backbone). P1. Amounts of reactants used: oEGMA = 409 mg, 0.861 mmol; BTAMA = 68.0 mg, 93.7 μmol; CTA = 2.85 mg, 10.2 μmol; AIBN = 0.33 mg, 2.0 μmol. Reaction time: 18 h. SEC: Mn = 25.8 kDa, Đ = 1.24. P2. Amounts of reactants used: oEGMA = 405 mg, 0.853 mmol; BTAMA = 67 mg, 93 μmol; CTA = 1.07 mg, 3.83 μmol; AIBN = 0.13 mg, 0.8 μmol. Reaction time: 23 h. SEC: Mn = 32.2 kDa, Đ = 1.23. P3. Amounts of reactants used: oEGMA = 911 mg, 1.92 mmol; BTAMA = 155 mg, 213 μmol; CTA = 1.494 mg, 5.354 μmol; AIBN = 0.18 mg, 1.1 μmol. Reaction time: 46 h. SEC: Mn = 53.1 kDa, Đ = 1.72. P4. Amounts of reactants used: oEGMA = 917 mg, 1.93 mmol; BTAMA = 155 mg, 213 μmol; CTA = 1.000 mg, 3.584 μmol; AIBN = 0.13 mg, 0.8 μmol. Reaction time: 46 h. SEC: Mn = 55.2 kDa, Đ = 1.86.

performed in Spectra/Por Dialysis membranes (Spectrum Laboratories) with a molecular weight cutoff of 6−8 kDa. DMF-SEC measurements were carried out in PL-GPC-50 plus from Polymer Laboratories (Varian Inc. Company) with refractive index detector working in DMF containing 10 mM LiBr at 50 °C (flow rate: 1 mL min−1) on a Shodex GPC-KD-804 column (exclusion limit =400 kDa.; 0.8 cm i.d. × 300 mm) or on a Shodex GPC-KD-805 column (exclusion limit =5000 kDa.; 0.8 cm i.d. × 300 mm) which were calibrated with poly(ethylene oxide) (PEO) samples (Polymer Laboratories). Dynamic light scattering measurements discussed in Table 1 were performed on a Malvern μV Zetasizer equipped with a 830 nm laser and a scattering angle of 90° at a temperature of 20 °C. Samples were prepared by filtering solutions through a 0.2 μm PVDFfilter (Whatman) into a fluorescence cell with a path length of 1 cm. Static light scattering measurements and dynamic light scattering measurements discussed in Figure 5 were conducted on an ALV/CGS3 MD-4 compact goniometer system equipped with a multiple tau digital real time correlator (ALV-7004, solid state laser: λ = 532 nm; 40 mW). Measurements were performed over an angular range between 20 and 150°, performing 3 × 30s acquisitions at each angle. The scattering vector q = (4πnD/λ)/sin(θ/2), where nD is the refractive index of the solvent, λ is the laser wavelength and θ is the scattering angle. The samples were prepared at a concentration of 1 to 100 mg mL−1 in Milli-Q water and measured without filtration in 5 mm borosilicate cells. Small angle neutron scattering (SANS) experiments were performed at the Institute Laue-Langevin (ILL) on the D11 instrument. Sample-to-detector distances of 1.2 and 8 m were used together with a neutron wavelength of 10 Å. The observed q range was 4 × 10−2 nm−1 ≤ q ≤ 3.0 nm−1. The 2D images were radially averaged to obtain the intensity I(q) vs q profiles and calibrated to absolute scale using Milli-Q water as a reference. Standard data reduction procedures, i.e. subtraction of the empty capillary and solvent contribution, were applied using the LAMP software package. The samples were prepared at a concentration of 1 mg mL−1 in mixtures of deuterated 2-propanol and D2O (0 ≤ ϕIPA ≤ 1) and held in 2 mm Hellma quartz cells. Small angle X-ray scattering (SAXS) was performed on a SAXSLAB Ganesha system with a GeniX-Cu ultralow divergence source producing X-ray photons with a wavelength of 1.54 Å and a flux of 1 × 108 ph/s. Scattering patterns were collected using a Pilatus 300 K silicon pixel detector. Sample-to-detector (SD) distances of 0.73 and 1.53 m were used giving an observed q range of 6.5 × 10−2 nm−1 ≤ q ≤ 4.5 nm−1. The 2D images were radially averaged to obtain the intensity I(q) vs q profiles and calibrated to absolute scale using the Saxsgui software package. The liquid samples were contained in 2 mm quartz capillaries. Typical measurement times were 3 h at a S-D distance of 0.73 m and 5 h at 1.53 m. Synthesis of Polymers P1−P4. In a Schlenck-tube, oEGMA, BTAMA (0.11 equiv to oEGMA), 4-cyano-4-methyl-5-(phenylthio)-5thioxopentanoic acid (CTA, 1/target DP equiv to monomers) and azobis(isobutyronitrile) (AIBN, 0.2 equiv to CTA) were dissolved in dioxane (4 mL). The mixture was subjected to three freeze−pump− thaw cycles, backfilled with argon and placed in a preheated oil-bath at 60 °C. After 18−46h the polymerization was stopped by placing the reactor in a liquid-nitrogen bath. Conversions were determined with

■

RESULTS Synthesis and Characterization of P1−P4. Random copolymers incorporating oligo(ethylene glycol)methacrylate (oEGMA with Mn = 475 and DP = 8.5−9) and BTA methacrylate (BTAMA) as the monomers were prepared using reversible addition−fragmentation chain transfer (RAFT) polymerizations (Scheme 2).28 Four water-soluble copolymers, P1−P4, with DPs ranging from 110 to 450, were prepared by a random copolymerization of oEGMA and BTAMA using 4cyano-4-methyl-5-(phenylthio)-5-thioxopentanoic acid as chain transfer agent (CTA) and azobis(isobutyronitrile) (AIBN) as radical source. In all cases, a loading of 10 mol % of BTAs in the feed was chosen, which is a compromise between polymer solubility and sufficient optical density for the spectroscopic studies (see below). P1−P4 were characterized by 1H NMR spectroscopy, size-exclusion chromatography (SEC), and dynamic light scattering (DLS). The conversion of methacrylate groups was determined from 1H NMR measurements on the crude reaction mixtures, while the percentage of BTAs incorporated in the final polymers was calculated from 1H NMR measurements on the purified, precipitated polymers. The results are presented in Table 1. 2949

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

Because of the low monomer concentration applied in the polymerizations (20 wt %) and the low ratio of AIBN to CTA (0.2 equiv), initiator-derived chains can be neglected.29 If we assume a 100% initiation efficiency for the RAFT CTA, the theoretical DP, DPth, can be calculated by multiplying the conversion with the target DP. The thus calculated DPth for P1−P4 ranged from 110 to 460. With SEC measurements (DMF, PEG standards) the number-average molecular weight (Mn) and molar mass dispersities (Đ) of P1−P4 were determined. The Mn of P1−P4 ranged from 25.8 kDa to 55.2 kDa while the Đ ranged from 1.24 to 1.86. The higher dispersities in P3 and P4 are indicative for the well-known limits of conventional RAFT-polymerizations.29 For all polymers, the SEC-traces showed a unimodal, Gaussian shaped peak, indicating that only one species is present in the polymer (see for example P2 in Figure 1A).

Figure 2. (A) Temperature-wavelength scan measured at a cooling rate of 60 K h−1 at λ = 223 nm for solutions of P1−P4 in water (cBTA = 50 μM, l = 0.5 cm); (B) Normalized cooling curves of the scans shown in panel A representing the fraction of helical bonds. The fraction of helical bonds, f h, was calculated from f h(T) = (θ(T) − θu)/(θf − θu)) where θ(T) is the measured ellipticity at temperature T; θu and θf are the ellipticities of the fully unfolded and fully folded state, respectively. The latter two values could be determined from the data.

completely overlapping, which is indicative for a system lacking hysteresis. Furthermore, the curves show a shallow shape and lack the sharp onset that is generally observed for cooperative intermolecular BTA self-assembly.30 From the melting curves obtained by CD, the fraction of helical bonds as a function of temperature was calculated for the four different chain lengths (Figure 2B). As can be observed from this figure, all curves are superimposable which is a strong indication that cooperative non-nearest neighbor interactions are absent in the folding of BTA-based SCPNs. As a result, the transition temperature Tm (defined as the temperature at which half of the BTAs that can aggregate in the polymer are aggregated) is identical for all polymers (around 50 °C). These results are in line with earlier results obtained for water-soluble BTA containing SCPNs, in which concentration-dependent CD-measurements showed a concentration-independent transition temperature Tm, even though the magnitude of the CD-effect was concentrationdependent.13a Shape and Size of P1−P4 after Folding in Water. Recently, we showed by small angle neutron scattering (SANS) that BTA containing copolymers with a DP ∼ 100 form ellipsoidal-shaped SCPNs in water at room temperature; while heating the solution resulted in smaller and more spherically shaped SCPNs.13d Here, we investigate how an increase in the DP affects the global conformation of the copolymers in water. Therefore, SANS experiments were performed on P1−P4 in D2O at 25 and 60 °C (cpolymer = 1 mg mL−1), corresponding to states wherein the BTA moieties are mostly aggregated or not aggregated, respectively. In addition, we measured SANS in 2propanol-d8 (IPA-d8) at 25 °C, a solvent that competes with BTA−BTA hydrogen bond formation and in which BTA aggregation is therefore absent. As an example, the SANS curves taken at 25 °C in D2O for P1−P4 are shown in Figure 3A. From these scattering curves, we extract the molecular weights (MSANS), which correspond reasonably well to the molecular weight determined by NMR (Mw,calc.) (Table 2; see the Supporting Infomation for details). This observation confirms the DLS results and indicates that, when BTAs are aggregated, the particles formed by P1−P4 in water consist of a single polymer chain only. In fact, upon decreasing and increasing the concentration of the polymer P1 in water to 0.5 and 2 g mL−1, respectively, the concentration normalized scattering profiles are superimposable, showing that

Figure 1. (A) Representative SEC-trace for P2 (DP = 140) in DMF + 10 mM LiBr. (B) Intensity distribution vs hydrodynamic diameter (Dh) for P2 measured at a 90° angle at 20 °C, averaged over 3 measurements for a 1 mg/mL solution in water.

DLS measurements were conducted on P1−P4 in water (cpolymer = 1 mg mL−1, see as example P2 in Figure 1B). The hydrodynamic radii (Rh) range from 6.1 nm for P1 to 28.9 nm for P4. Comparison of the values for Rh measured for P1−P4 with values obtained from literature for water-soluble BTAcontaining SCPNs, suggests that P1−P4 form polymeric nanoparticles in aqueous solutions that predominantly consist of one single polymer chain only.13a,d Folding of P1−P4 in Water Followed by CD Spectroscopy. The folding behavior of P1−P4 was investigated by temperature-dependent CD-measurements on dilute aqueous solutions. The total concentration of BTAs (cBTA) was kept constant at 50 μM (cpolymer ∼ 0.3 mg mL−1) for all polymers, while the temperature was varied from 85 to 0 °C at a cooling rate of 60 K h−1. The CD-effect was probed at λ = 223 nm, the maximum of the CD-effect of BTAs with a stereogenic center on the odd-position of the aliphatic side-chain.21c,d The individual CD-spectra show a sign and a shape that is in line with literature values for BTAs with S-chirality and the stereogenic center on the 3-position of the aliphatic side chain (Figure 2A).21 To compare the maximum CD-effect of P1−P4 with known values, we quantified the CD results by calculating the molar circular dichroism, Δε. The values calculated for Δε vary between −13 and −19 L mol−1 cm−1 at 0 °C. These absolute numbers are substantially lower than those of enantiomerically pure free BTAs in water (|Δε| = 40 L mol−1 cm−1).21f The temperature-dependent CD curves of P1−P4 are highly similar (Figure 2A). Subsequent heating and cooling runs are 2950

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

Figure 3. SANS profiles for (A) P1−P4 in D2O at 25 °C and cpolymer = 1 mg mL−1. (B) Concentration normalized profiles for P1 at cpolymer = 0.5, 1, and 2.5 mg mL−1 in D2O at 25 °C.

Figure 4. (A) RG,IFT for P1−P4 as a function of solvent composition and temperature at cpolymer = 1 mg mL−1. (B) Cross-sectional radius (R) and major radius (νR) of P1−P4 as a function of solvent composition and temperature at cpolymer = 1 mg mL−1.

Table 2. Results of the SANS Measurements on P1−P4 polymer

I0a [cm−1]

MSANSb [kDa]

Mw,calcc [kDa]

P1 P2 P3 P4

0.613 0.774 1.207 2.750d

103 129 202 460

68.6 85.3 277.9 429.0

BTA structure. Similarly upon changing solvent from water to 2-propanol we observe a decrease in νR and the particle dimensions (νR or RG,IFT) adopt values in between that of the extremes found in aqueous solution. Effect of Polymer Concentration on SCPN Character. Application of SCPNs as for example coating materials or catalyst particles requires solutions containing significantly higher concentrations of particles than the typically investigated 1 mg mL−1.13a,c To investigate how the total polymer concentration influences the single chain character of the formed particles, we performed dynamic and static light scattering (DLS and SLS) and small-angle X-ray scattering (SAXS) experiments on P3 in H2O at 1, 10, and 100 mg mL−1. Remarkably, even at a polymer concentration of 100 mg mL−1, the viscosity of the solution does not significantly increase. In DLS experiments, the correlation functions of P3 clearly show the presence of two relaxation processes at concentrations of 10 and 100 mg mL−1 (Figure 5A). Both processes are consistent with a diffusive mode, as indicated by the q2 dependence of the extracted decay rates (Γ) shown in Figure 5B. Over the entire concentration range, the fast process has a diffusion constant (D) of 3.35 × 10−11 m2 s−1 corresponding to an RH of 7.3 nm. The slow process slows down upon an increase in polymer concentration; we compute a diffusion constant of 3.9 × 10−12 m2 s−1 and 3.2 × 10−13 m2 s−1 at 1 and 100 mg mL−1, respectively (corresponding to a RH of 63 and 770 nm) (Figure 5C). While the fast process corresponds to translational diffusion of SCPNs, we attribute the slow process to translational diffusion of a multichain polymeric nanoparticle (MCPN), which is an agglomerate of a number of polymer chains. We can estimate the relative ratio of SCPN to agglomerates by analyzing their relative contributions to the total forward scattered intensity (see Supporting Infomation for details). This results in number ratios of SCPNs to MCPNs of 1 × 106, 5 × 105 and 7 × 104 to 1 (for 1, 10, and 100 mg mL−1, respectively). Thus, although the content of MCPNs increases

Forward scattered intensity (I0) - taken as the average intensity at q ≤ 0.07 nm−1. bMolecular weights determined by SANS (MSANS) of P1− P4 in D2O at 25 °C (cpolymer = 1 mg mL−1). cCalculated molecular weight (Mw,calc, based on Mmonomer × DPn × Đ). dEstimated from lowest q data point. a

interparticle interactions are indeed negligibly small in this concentration range (Figure 3B). Next, we studied the size and shape of P1−P4 in three conditions: unfolded polymers in pure 2-propanol at 25 °C, partially unfolded polymers in pure water at 60 °C and folded polymers in pure water at 25 °C. We determined the radius of gyration (RG,IFT) of the SCPN by indirect Fourier transform (IFT) analysis. In addition, we fit the scattering profiles to a model for an ellipsoidal particle which gives values for the cross-sectional radius and an aspect ratio (Table 3; see Supporting Infomation for details). From the aspect ratio (ν) and the cross-sectional radius (R), the major radius (νR, corresponding to ν × R) can be calculated. Both RG,IFT and the major radius obtained in the ellipsoidal model fit strongly depend on the degree of polymerization (Figure 4, parts A and B).31 By contrast, the cross-sectional radius (R) does not vary with DP and remains constant at ca. 3 nm. Hence, upon extension of the polymer chains, by the addition of more segments to the polymer backbone, the polymer particles seem to preferentially expand in one direction only. The influence of solvent and temperature on the conformation of the SCPN is equivalent to that observed in our previous study of a lower DP SCPN.13d In pure water, elevating the temperature from 25 to 60 °C, we find a decrease in particle elongation (νR), i.e. the SCPN adopts a more globular shape upon the loss of internal

Table 3. Cross-Sectional Radius (R) and Aspect Ratio (ν) from Ellipsoidal Particle Fit and RG,IFT from IFT of P1−P4 as a Function of Solvent and Temperature at cpolymer =1 mg mL−1 ν [−]

R [nm]

RG,IFT [nm]

polymer

25 °C D2O

60 °C D2O

25 °C IPA-d8

25 °C D2O

60 °C D2O

25 °C IPA-d8

25 °C D2O

60 °C D2O

25 °C IPA-d8

P1 P2 P3 P4

2.8 2.9 3.2 3.3

3.3 3.3 3.5 3.6

2.7 2.8 3.3 3.6

6.3 7.5 7.1 13.0

2.7 3.1 5.2 8.6

4.9 5.3 7.8 11.4

7.6 8.7 10.1 17.8

4.5 5.1 8.9 11.3

6.1 7.0 10.0 12.3

2951

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

Figure 5. Dynamic light scattering experiments on P3 in H2O at cpolymer = 1, 10, and 100 mg mL−1 and 25 °C: (A) Correlation functions at an angle of 30° (gray) and 150° (black). (B) Angular dependence of the fast (black) and slow (gray) diffusion constants. (C) Concentration dependence of diffusion constant of fast (Dfast) and slow (Dslow) process.

temperature, indicating that the degree of polymerization does not affect the degree of aggregation of BTA units attached to the polymer backbone. However, the values for |Δε| are a factor of 2−2.5 smaller than expected for a system in which all BTA units are self-assembled, which indicates that not all BTAs are aggregated when attached to a polymer chain or that the preferred helicity is not fully expressed in these systems. This may be due to a competition between the solvent and/or the ethylene glycol grafts and BTA self-assembly33 or a limit to the extent that BTAs can aggregate because of steric constraints relayed by the polymer’s conformational preferences. Remarkably, the CD cooling curves of P1−P4 are superimposable, indicating that the length of the polymers does not affect the melting curves. As a result, the Tm of the polymer is independent of the DP of the polymer, a clear indication that non-neighboring interactions between the pendent BTA units are absent.23b,25−27 The apparent lack of cooperativity in the intramolecular folding of poly(oEGMA-co-BTAMA) is remarkable since intermolecular BTA self-assembly is highly cooperative.34 SANS experiments reveal that an increase in DP results in a constant cross-section R of the SCPN while the major radius νR increases almost linearly. Upon increasing the DP of a polymer, the BTA units in an added polymer section would not be in the proximity of those already present in the previous sections of the polymer, leaving the “effective” BTA concentration constant. Thus, added sections of polymer would act as independently folding units, giving a length independent folding behavior. This proposal is schematically depicted in Figure 7. The cause of this elongation behavior might be found in considering the entropic penalty of the polymeric backbone to fold around a BTA-stack. Thus, the entropic penalty of the folding of the polymeric backbone in a “locked” conformation is offsetting the normal cooperative behavior of BTAs which has an enthalpic origin. This

upon increasing concentration, even at very high concentrations, the majority of the particles contain single polymer chains only. More insight into the nature of the solution of P3 at these elevated concentrations was obtained from combining SLS and SAXS experiments (Figure 6A). At a concentration of 1 mg

Figure 6. (A) Combined SAXS and SLS profiles for P3 in H2O at 1, 10, and 100 mg mL−1 at 25 °C (SLS rescaled to align with 1 mg mL −1 SAXS profile). (B) Extracted structure factor S(q) for P3 in H2O at 10 and 100 mg mL−1 at 25 °C.

mL−1, a q independent scattered intensity is observed at low q in both SAXS and SLS, as expected for single-chain particles in solution. Upon increasing the concentration to 10 mg mL−1, we observe a similar scattering profile with an approximately 10fold increase in intensity and an increase in scattered intensity in the low q regime. Upon a further increase in concentration to 100 mg mL−1, the total scattered intensity does not increase 10fold and the low q upturn is more clearly observable, suggesting that interparticle interactions can no longer be ignored at these high concentrations. Structure factors were determined for the 10 and 100 mg mL−1 solutions from the ratio between the high and low concentration scattering profiles (Figure 6B; see Supporting Infomation for details). The obtained profile, namely a depression of the scattered intensity at intermediate q values followed by an upturn in scattered intensity at low q values, resembles structure factors measured in various colloidal dispersions displaying attractive hard-sphere behavior. The latter indicates that SCPNs are mutually attractive.32

■

DISCUSSION The normalized CD spectra of poly(oEGMA-co-BTAMA) (P1−P4) are superimposable at cBTA = 50 μM at every

Figure 7. Tentative picture of a SCPN based on P3 at 25 °C in H2O. The drawn ellipsoid has the aspect ratio determined from the SANS profile under these conditions. 2952

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

to a larger concentration range and investigating, for example, the effect of temperature on the interparticle interactions are important to better understand SCPNs and their potential applications.

phenomenon has been observed in more systems, such as in the formation of supramolecular naphthalenediimide nanotubes.35 This hypothesis could also explain why a rather low molar circular dichroism is observed for all polymers. Increasing the total polymer concentration reveals the attractive interactions between particles. The onset of the appearance of a pronounced structure factor at approximately 100 mg mL−1 can be rationalized as follows. By calculating the mean particle distance in solution, we obtain values of 98, 46, and 22 nm at 1, 10, and 100 mg mL−1, respectively (using a molecular weight of 300 kDa; see Supporting Infomation for details). In other words, an increase in the polymer concentration reduces the average interparticle distance down to values in the order of the actual particle size of RH = 7.3 nm at 100 mg mL−1. At such close interparticle distances, BTA− BTA interactions between particles will start to play a more important role. It is well-known that attractive interactions between “sticky” polymers−such as telechelic polymers with interacting groups placed at both chain ends−play a significant role only at distances close to interpolymer contact.36 Nevertheless, up to high polymer concentration a significant fraction of the particles is still present as a SCPN and no significant change in viscosity of the solution is observed. These observations are particularly interesting for applications of SCPNs. The single chain character even at high concentrations is important in applications such as catalytically active SCPNs. Here, one works at elevated SCPN concentrations while the compartmentalization induced by the SCPN is of crucial importance for efficient and selective catalysis. Furthermore, the low viscosity at high concentration facilitates handling of SCPN solutions. This would be particularly advantageous in, for example, coating applications where higher concentrations of particles would be required and low viscosities make spreading of a particle solution more convenient.

■

ASSOCIATED CONTENT

S Supporting Information *

Size-exclusion chromatography, 1H NMR spectroscopy, small angle neutron scattering, light scattering, and small angle X-ray scattering. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*(A.R.A.P) E-mail: [email protected]. *(I.K.V.) E-mail: [email protected]. Author Contributions §

These authors contributed equally to the work

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is financed by The Netherlands Organisation for Scientific Research (NWO - TOP grant: 10007851), the Dutch Ministry of Education, Culture and Science (Gravity program 024.001.035), the European Research Council (FP7/20072013, ERC Grant Agreement 246829) and NRSC-C. T.F.A.d.G. acknowledges financial support from The Netherlands Organization for Scientific research (NWO−VENI Grant 722.012.0001). I.K.V. is grateful for financial support from The Netherlands Organization for Scientific research (NWO−VENI Grant 700.10.406) and the European Union through the Marie Curie 5 Fellowship program FP7-PEOPLE-2011-CIG (Contract No. 293788). We gratefully thank Peter A. Korevaar (Eindhoven University of Technology) for inspiring discussions. The ICMS Animation Studio (Eindhoven University of Technology) is acknowledged for providing the artwork.

■

CONCLUSIONS We performed a detailed conformational study on random copolymers of methacrylate monomers bearing side chains comprising oligo(ethylene glycol) and chiral benzene-1,3,5tricarboxamide, by making use of scattering methods in combination with spectroscopy. By means of small angle scattering we have unambiguously shown that poly(oEGMA-coBTAMA) with 10% BTA loading folds in water into a SCPN consisting of a single polymer chain. Circular dichroism spectroscopy reveals the helical BTA structure in the interior of the SCPN. At room temperature in pure water the SCPNs are best described as elongated objects. Increasing the DP results in a constant cross-section of the particles but an increasing aspect ratio, indicating that the polymers adopt highly stretched conformations. When increasing the temperaturethereby reducing the strength of the BTA hydrogen bondinga decrease in the size and the aspect ratio of the SCPN is observed. Detailed conformational studies on SCPN systems are crucial to further develop the field. The elongation induced by (directional) secondary interactions (hydrogen-bonding, π−π interactions and solvophobic effects) may prove to be highly advantageous to fine-tune the functionality of SCPNs in a variety of applications. These elongated conformations presumably result in a noncooperative folding behavior. Moreover our initial concentration-dependent experiments show that these polymers persist mainly as SCPNs up to concentrations of 100 mg mL−1. Expanding these experiments

■

REFERENCES

(1) (a) Aiertza, M. K.; Odriozola, I.; Cabañero, G.; Grande, H.-J.; Loinaz, I. Cell. Mol. Life. Sci. 2012, 69, 337−346. (b) Altintas, O.; Barner-Kowollik, C. Macromol. Rapid Commun. 2012, 33, 958−971. (c) Ouchi, M.; Badi, N.; Lutz, J.-F.; Sawamoto, M. Nat. Chem. 2011, 3, 917−924. (2) Beck, J. B.; Killops, K. L.; Kang, T.; Sivanandan, K.; Bayles, A.; Mackay, M. E.; Wooley, K. L.; Hawker, C. J. Macromolecules 2009, 42, 5629−5635. (3) (a) Zhu, B.; Ma, J.; Li, Z.; Hou, J.; Cheng, X.; Qian, G.; Liu, P.; Hu, A. J. Mater. Chem. 2011, 21, 2679−2683. (b) Zhu, B.; Qian, G.; Xiao, Y.; Deng, S.; Wang, M.; Hu, A. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 5330−5338. (4) (a) Harth, E.; van Horn, B.; Lee, V. Y.; Germack, D. S.; Gonzales, C. P.; Miller, R. D.; Hawker, C. J. J. Am. Chem. Soc. 2002, 124, 8653− 8660. (b) Pyun, J.; Tang, C.; Kowalewski, T.; Fréchet, J. M. J.; Hawker, C. J. Macromolecules 2005, 38, 2674−2685. (5) He, J.; Tremblay, L.; Lacelle, S.; Zhao, Y. Soft Matter 2011, 7, 2380−2386. (6) Croce, T. A.; Hamilton, S. K.; Chen, M. L.; Muchalski, H.; Harth, E. Macromolecules 2007, 40, 6028−6031. (7) (a) Ormategui, N.; García, I.; Padro, D.; Cabañero, G.; Grande, H. J.; Loinaz, I. Soft Matter 2012, 8, 734−740. (b) Ruiz de Luzuriaga, A.; Ormategui, N.; Grande, H. J.; Odriozola, I.; Pomposo, J. A.; Loinaz, I. Macromol. Rapid Commun. 2008, 29, 1156−1160. (c) Schmidt, B. V. 2953

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954

Macromolecules

Article

K. J.; Fechler, N.; Falkenhagen, J.; Lutz, J.-F. Nat. Chem. 2011, 3, 234− 238. (8) Sanchez-Sanchez, A.; Asenjo-Sanz, I.; Buruaga, L.; Pomposo, J. A. Macromol. Rapid Commun. 2012, 33, 1262−1267. (9) (a) Cherian, A. E.; Sun, F. C.; Sheiko, S. S.; Coates, G. W. J. Am. Chem. Soc. 2007, 129, 11350−11351. (b) Mecerreyes, D.; Lee, V.; Hawker, C. J.; Hedrick, J. L.; Wursch, A.; Volksen, W.; Magbitang, T.; Huang, E.; Miller, R. D. Adv. Mater. 2001, 13, 204−208. (c) Moreno, A. J.; Lo Verso, F.; Sanchez-Sanchez, A.; Arbe, A.; Colmenero, J.; Pomposo, J. A. Macromolecules 2013, 46, 9748−9759. (d) PerezBaena, I.; Barroso-Bujans, F.; Gasser, U.; Arbe, A.; Moreno, A. J.; Colmenero, J.; Pomposo, J. A. ACS Macro Lett. 2013, 2, 775−779. (10) Tuten, B. T.; Chao, D.; Lyon, C. K.; Berda, E. B. Polym. Chem. 2012, 3, 3068−3071. (11) (a) Murray, B. S.; Fulton, D. A. Macromolecules 2011, 44, 7242− 7252. (b) Whitaker, D. E.; Mahon, C. S.; Fulton, D. A. Angew. Chem., Int. Ed. 2013, 52, 956−959. (c) Sanchez-Sanchez, A.; Fulton, D. A.; Pomposo, J. A. Chem. Commun. 2014, 50, 1781−1874. (d) Frank, O. G.; Tuten, B. T.; Prasher, A.; Chao, D.; Berda, E. B. Macromol. Rapid Commun. 2014, 35, 249−253. (12) Seo, M.; Beck, J. B.; Paulusse, J. M. J.; Hawker, C. J.; Kim, S. Y. Macromolecules 2008, 41, 6413−6418. (13) (a) Terashima, T.; Mes, T.; de Greef, T. F. A.; Gillissen, M. A. J.; Besenius, P.; Palmans, A. R. A.; Meijer, E. W. J. Am. Chem. Soc. 2011, 133, 4742−4745. (b) Mes, T.; van der Weegen, R.; Palmans, A. R. A.; Meijer, E. W. Angew. Chem., Int. Ed. 2011, 50, 5085−5089. (c) Huerta, E.; Stals, P. J. M.; Meijer, E. W.; Palmans, A. R. A. Angew. Chem., Int. Ed. 2013, 52, 2906−2910. (d) Gillissen, M. A. J.; Terashima, T.; Meijer, E. W.; Palmans, A. R. A.; Voets, I. K. Macromolecules 2013, 46, 4120−4125. (e) Artar, M.; Terashima, T.; Sawamoto, M.; Meijer, E. W.; Palmans, A. R. A. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 12−20. (14) (a) Foster, E. J.; Berda, E. B.; Meijer, E. W. J. Am. Chem. Soc. 2009, 131, 6964−6966. (b) Berda, E. B.; Foster, E. J.; Meijer, E. W. Macromolecules 2010, 43, 1430−1437. (c) Stals, P. J. M.; Gillissen, M. A. J.; Nicolaÿ, R.; Palmans, A. R. A.; Meijer, E. W. Polym. Chem. 2013, 4, 2584−2597. (d) Foster, E. J.; Berda, E. B.; Meijer, E. W. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 118−126. (e) Stals, P. J. M.; Li, Y.; Burdyńska, J.; Nicolaÿ, R.; Palmans, A. R. A.; Meijer, E. W.; Matyjaszewski, K.; Sheiko, S. S. J. Am. Chem. Soc. 2013, 135, 11421−11424. (15) Hosono, N.; Gillissen, M. A. J.; Li, Y.; Sheiko, S. S.; Palmans, A. R. A.; Meijer, E. W. J. Am. Chem. Soc. 2013, 135, 501−510. (16) Gillissen, M. A. J.; Voets, I. K.; Meijer, E. W.; Palmans, A. R. A. Polym. Chem. 2012, 3, 3166−3174. (17) (a) Appel, E. A.; Dyson, J.; del Barrio, J.; Walsh, Z.; Scherman, O. A. Angew. Chem., Int. Ed. 2012, 51, 4185−4189. (b) Appel, E. A.; del Barrio, J.; Dyson, J.; Isaacs, L.; Scherman, O. A. Chem. Sci. 2012, 3, 2278−2281. (18) Altintas, O.; Rudolph, T.; Barner-Kowollik, C. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 2566−2576. (19) (a) Altintas, O.; Gerstel, P.; Dingenouts, N.; Barner-Kowollik, C. Chem. Commun. 2010, 46, 6291−6293. (b) Altintas, O.; Lejeune, E.; Gerstel, P.; Barner-Kowollik, C. Polym. Chem. 2012, 3, 640−651. (20) Terashima, T.; Sugita, T.; Fukae, K.; Sawamoto, M. Macromolecules 2014, 47, 589−600. (21) (a) Cantekin, S.; de Greef, T. F. A.; Palmans, A. R. A. Chem. Soc. Rev. 2012, 41, 6125−6137. (b) Smulders, M. M. J.; Schenning, A. P. H. J.; Meijer, E. W. J. Am. Chem. Soc. 2008, 130, 606−611. (c) Stals, P. J. M.; Smulders, M. M. J.; Martín-Rapún, R.; Palmans, A. R. A.; Meijer, E. W. Chem.Eur. J. 2009, 15, 2071−2080. (d) Nakano, Y.; Hirose, T.; Stals, P. J. M.; Meijer, E. W.; Palmans, A. R. A. Chem. Sci. 2012, 3, 148−155. (e) Besenius, P.; Portale, G.; Bomans, P. H. H.; Janssen, H. M.; Palmans, A. R. A.; Meijer, E. W. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 17888−17893. (f) Leenders, C. M. A.; Albertazzi, L.; Mes, T.; Koenigs, M. M. E.; Palmans, A. R. A.; Meijer, E. W. Chem. Commun. 2013, 49, 1963−1965. (22) Rose, G. D.; Fleming, P. J.; Banavar, J. R.; Marita, A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16623−16633.

(23) (a) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101, 3893−4011. (b) Prince, R. B.; Saven, J. G.; Wolynes, P. G.; Moore, J. S. J. Am. Chem. Soc. 1999, 121, 3114−3121. (c) Wackerly, J. W.; Moore, J. S. Macromolecules 2006, 39, 7269−7276. (24) Cornelissen, J. J. L. M.; Donners, J. J. J. M.; de Gelder, R.; Graswinckel, W. S.; Metselaar, G. A.; Rowan, A. E.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 2001, 293, 676−680. (25) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959, 31, 526−535. (26) Lifson, S.; Roig, A. J. Chem. Phys. 1961, 34, 1963−1974. (27) Eisenberg, H.; Felsenfeld, G. J. Mol. Biol. 1967, 30, 17−37. (28) Moad, G.; Rizzardo, E.; Thang, S. H. Acc. Chem. Res. 2008, 41, 1133−1142. (29) (a) Rizzardo, E.; Moad, G.; Thang, S. H. in Handbook of RAFTpolymerization; Barner-Kowollik, C., Ed.; Wiley-VCH: Weinheim, 2008; p 189−234. (b) Rizzardo, E.; Moad, G.; Thang, S. H. Aust. J. Chem. 2005, 58, 379−410. (30) Smulders, M. M. J.; Nieuwenhuizen, M. M. L.; de Greef, T. F. A.; van der Schoot, P.; Schenning, A. P. H. J.; Meijer, E. W. Chem. Eur. J. 2010, 16, 362−367. (31) The near-linear dependence of RG,IFT and νR on DP indicates that the high molecular weight polymers behave like rodlike structures, while the results of a previous scattering studyusing form factor modelling of the full scattering profilesrevealed that the persistence length of a similar poly(oEGMA-co-BTAMA) polymer with DP = 100 and 10 mol% BTAs is shorter than its contour length (see ref 13d). We attribute this apparent contradiction to the relatively low molecular weight of the SCPN. Experimentally determined scaling exponents α (with RG ∝ Mwα) differ from predicted scaling exponents 0.5 and 0.588, for polymer conformations following random walk and self-avoiding walk statistics, at low Mw (which is for example Mw < 104 Da for polystyrene in a Θ solvent). (32) Salgi, P.; Rajagopalan, R. Adv. Colloid Interface Sci. 1993, 43, 169−288. (33) de Greef, T. F. A.; Nieuwenhuizen, M. M. L.; Stals, P. J. M.; Fitié, C. F. C.; Palmans, A. R. A.; Sijbesma, R. P.; Meijer, E. W. Chem. Commun. 2008, 4306−4308. (34) Filot, I. A. W.; Palmans, A. R. A.; Hilbers, P. A. J.; van Santen, R. A.; Pidko, E. A.; de Greef, T. F. A. J. Phys. Chem. B 2010, 114, 13667− 13674. (35) Ponnuswamy, N.; Dan Pantoş, G.; Smulders, M. M. J.; Sanders, J. K. M. J. Am. Chem. Soc. 2012, 134, 566−573. (36) Lemmers, M.; Voets, I. K.; Cohen Stuart, M. A.; v. der Gucht, J. Soft Matter 2011, 7, 1378−1389.

2954

dx.doi.org/10.1021/ma500273g | Macromolecules 2014, 47, 2947−2954